Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice

Research article Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice

Angélica M. Santos,1 Jason Jung,2 Nazneen Aziz,3 Joseph L. Kissil,1 and Ellen Puré1,4

1The Wistar Institute, Philadelphia, Pennsylvania, USA. 2Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 3Point Therapeutics Inc., Boston, Massachusetts, USA. 4Ludwig Institute for Cancer Research, Philadelphia, Pennsylvania, USA.

Membrane-bound proteases have recently emerged as critical mediators of tumorigenesis, angiogenesis, and metastasis. However, the mechanisms by which they regulate these processes remain unknown. As the cell surface serine protease fibroblast activation protein (FAP) is selectively expressed on tumor-associated fibro-blasts and pericytes in epithelial tumors, we set out to investigate the role of FAP in mouse models of epithelial-derived solid tumors. In this study, we demonstrate that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in both an endogenous mouse model of lung cancer driven by the K-ras G12D mutant and a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice. Interestingly, growth of only the K-ras G12D–driven lung tumors was also attenuated by inhibition of the closely related protease dipeptidyl peptidase IV (DPPIV). Our results indicate that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofi-broblast content, and decreases blood vessel density in tumors. These data provide proof of principle that targeting stromal cell–mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.

Introduction

Tumors?are?composed?of?heterogeneous?populations?of?cells,?including?transformed?cells?and?a?multitude?of?untransformed?cells.?Although?the?prevalence?of?different?cell?types?varies?among?tumors?and?at?different?stages?of?tumor?progression,?they?include?infiltrating?inflammatory?and?immune?cells,?endothelial?cells?and?mesenchymal-derived?smooth?muscle?cells,?pericytes,?and?tumor-associated?fibroblasts?(TAFs),?which?are?referred?to?here-in?collectively?as?stromal?cells.?TAFs?are?a?heterogeneous?popu-lation?that?can?be?phenotypically?distinguished?from?normal?fibroblasts.?Fibroblast?activation?protein?(FAP)?has?emerged?as?a?marker?of?reactive?fibroblasts?in?tumors?as?well?as?granulation?tissue?and?in?fibrotic?lesions.?Although?the?phenotypic?and?func-tional?heterogeneity?among?TAFs?is?yet?to?be?fully?explored,?at?least?a?subset?of?TAFs?have?been?characterized?as?myofibroblasts,?based?on?expression?of?αSMA.

Stromal?cells?communicate?among?themselves?as?well?as?with?cancer?cells?and?inflammatory?and?immune?cells?directly?through?cell?contact?and?indirectly?through?paracrine/exocrine?signaling,?proteases,?and?modulation?of?the?ECM.?This?complex?communica-tions?network?is?pivotal?to?providing?the?appropriate?microenvi-ronment?to?support?tumorigenesis,?angiogenesis,?and?metastasis?(1,?2).?Considering?the?key?role?of?the?microenvironment?in?tumor?development,?identification?of?stromal?targets?for?cancer?thera-peutics?is?of?great?interest?and?could?provide?strategies?that?will?complement?therapies?directed?against?cancer?cells.?Among?these?potential?targets?is?an?array?of?proteases?(3).

Proteases?are?important?factors?in?the?pathophysiology?of?tumors,?having?requisite?roles?in?angiogenesis?and?metastasis.?The?major?classes?of?endopeptidases?involved?in?ECM?degradation?include?serine?(such?as?the?plasminogen?activator,?uPA),?cysteine?(cathepsins),?aspartyl,?and?MMPs?(4,?5).?Numerous?reports?have?demonstrated?increased?expression?of?ECM?degrading?enzymes,?including?type?IV?collagenase?(MMP-2),?cathepsin?B,?cathepsin?D,?and?serine?proteases,?such?as?uPA?in?tumor?cells?(6),?but?the?benefit?of?targeting?the?activity?of?proteases?in?cancer?has?yet?to?be?estab-lished.?Also,?the?widespread?expression?of?many?of?these?enzymes?is?likely?to?limit?their?potential?as?therapeutic?targets.?In?contrast,?FAP?(also?called?FAPα?or?seprase)?has?recently?gained?attention?as?a?potential?target,?due?to?its?tightly?regulated?expression?in?the?tumor?stroma?and?structurally?defined?proteolytic?activity?(7–11);?however,?its?function?in?tumors?is?largely?unknown.

FAP?is?a?type?II?transmembrane?cell?surface?protein?belonging?to?the?post-proline?dipeptidyl?aminopeptidase?family,?sharing?the?highest?similarity?with?dipeptidyl?peptidase?IV?(DPPIV/CD26).?FAP?is?expressed?selectively?by?TAFs?and?pericytes?in?more?than?90%?of?human?epithelial?cancers?examined?(12–16).?It?is?also?expressed?during?embryonic?development?(17),?in?tissues?of?heal-ing?wounds?(18),?and?in?chronic?inflammatory?and?fibrotic?con-ditions?such?as?liver?cirrhosis?(19,?20)?and?idiopathic?pulmonary?fibrosis?(21),?as?well?as?on?bone?and?soft?tissue?sarcomas?(16,?22)?and?some?melanoma?(23).?Expression?of?FAP?is?not?however?detect-ed?in?benign?lesions?or?normal?adult?tissues?(23,?24),?while?DPPIV?is?more?widely?expressed?in?a?variety?of?cell?types?(reviewed?in?refs.?25,?26).?In?vitro?studies?have?shown?that?FAP?has?both?dipeptidyl?peptidase?(19,?24)?and?endopeptidase?activity?(8,?10,?27),?includ-ing?a?collagenolytic?activity?capable?of?degrading?gelatin?(28,?29)?and?type?I?collagen?(27,?30),?but?its?in?vivo?substrate(s)?is?yet?to?be?defined.?Based?on?the?highly?regulated?expression?and?restricted?distribution?of?FAP,?it?has?been?suggested?that?FAP?inhibition?may?be?useful?in?cancer?therapeutics.

Although?some?experimental?evidence?suggesting?FAP?pro-motes?tumorigenesis?has?been?reported,?the?models?used?in?these?studies?largely?failed?to?recapitulate?the?potentially?impor-tant?interactions?between?mesenchymal-derived?stromal?cells,?

Authorship note:?Joseph?L.?Kissil?and?Ellen?Puré?contributed?equally?to?this?work. Conflict of interest:?Ellen?Puré?received?research?support?in?2007?from?Point?Thera-peutics?Inc.

Citation for this article:?J. Clin. Invest.?119:3613–3625?(2009).?doi:10.1172/JCI38988.

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tumor?cells?and?immune?and?inflammatory?cells?that?typically?occur?in?tumors.?For?example,?a?number?of?these?studies?used?xenografts?of?human?tumor?cells?ectopically?overexpressing?FAP,?or?an?enzymatically?inactive?mutant,?transplanted?into?immune?incompetent?mice?(31,?32)?despite?the?fact?that?in?pri-mary?tumors,?FAP?is?expressed?only?by?the?TAFs?and?pericytes?and?not?by?tumor?cells.?Furthermore,?the?mechanisms?involved?were?not?defined.?To?determine?whether?FAP?promotes?tumori-genesis?and?understand?the?molecular?mechanism?by?which?this?might?occur?under?more?relevant?pathophysiologic?conditions,?we?studied?the?impact?of?genetic?deletion?of?FAP?and?pharma-cologic?inhibition?of?its?enzymatic?activity?in?both?syngeneic?transplant?and?endogenous?mouse?tumor?models?(33–37).?We?herein?demonstrate?that?endogenous?FAP?expressed?on?tumor?stromal?cells?promotes?tumor?progression?via?its?enzymatic?activ-ity.?Our?results?indicate?that?FAP?is?a?major?source?or?regulator?of?collagenase?activity?in?tumors,?FAP?promotes?tumor?growth?by?indirectly?promoting?tumor?cell?proliferation,?and?that?FAP?is?required?for?tumor?stromagenesis?and?vascularization.?In?addi-tion?to?providing?insight?into?the?mechanisms?by?which?FAP?pro-motes?tumorigenesis,?these?results?suggest?that?inhibition?of?FAP?enzymatic?activity?warrants?further?investigation?as?a?potential?therapeutic?approach.Results

Development of endogenous lung tumors is inhibited in FAP-deficient mice.?We?employed?a?K-ras G12D–driven?endogenous?model?of?lung?adenocarcinoma?to?examine?the?consequences?of?the?loss?of?FAP?on?tumorigenesis.?In?this?model,?lung?tumorigenesis?is?driven?by?the?conditional?activation?of?an?oncogenic?allele?of?K-ras G12D.?Acti-vation?of?the?K-ras G12D?allele?is?achieved?by?delivery?of?adenovirus?expressing?the?Cre?recombinase?(Ad-Cre),?resulting?in?recom-bination?and?removal?of?a?transcriptional?STOP?element?in?the?Lox-Stop-Lox?(LSL)?cassette?(34).?Fap-null?mice,?generated?by?LacZ?knockin,?are?viable?(17),?with?normal?lung?histology?(Supplemen-tal?Figure?1A;?supplemental?material?available?online?with?this?article;?doi:10.1172/JCI38988DS1)?and?similar?basal?collagen?content?to?littermate?Fap+/+?control?mice?(Supplemental?Figure?1B).?The?overall?morphology?of?lungs?from?LSL–K-ras G12D;Fap+/+,?LSL–K-ras G12D;Fap+/LacZ,?and?LSL–K-ras G12D;Fap LacZ/LacZ?littermates?in?the?absence?of?Ad-Cre?was?similar?(data?not?shown).?Ad-Cre?induced?tumors?in?all?genotypes,?but?reduced?tumor?burden?in?the?absence?of?FAP?was?evident?upon?macroscopic?inspection?of?lungs?(Supplemental?Figure?1C).?By?8?weeks?after?Ad-Cre?instillation (2.5?×?107?PFU/mouse),?extensive?epithelial?hyperplasia?of?the?alve-olar?region?(11?of?11?mice),?adenomas?(11?of?11?mice),?and?inciden-tal?pneumocyte?hyperplasia?(2?of?11?mice)?were?already?evident?in?

Figure 1

Development of lung tumors in LSL–K-

ras G12D;Fap+/+, LSL–K-ras G12D;Fap+/LacZ, and

LSL–K-ras G12D;Fap LacZ/LacZ mice. (A) Rep-

resentative sections from each genotype

at 8 weeks after Ad-Cre infection. Regions

of hyperplasia (asterisks) and adenomas

(pound symbols) are indicated. Original

magnification, ×4 (top row); ×40 (bottom 2

rows). Scale bar: 100 μm. Images that dis-

play areas of Ki67 staining, shown in the

bottom row of panels, were selected based

on similarity of tumor content (indicated by

solid lines) between genotypes, whereas

H&E-stained sections show randomly

selected representative areas, unrelated

to those shown for Ki67. (B) Tumor-to-lung

area (T/L) ratio in LSL–K-ras G12D;Fap+/+,

LSL–K-ras G12D;Fap+/LacZ, and LSL–K-ras G12D;

Fap LacZ/LacZ mice at 8 weeks after Ad-Cre

infection (n = 11) and proliferative index,

which is calculated as percentage of Ki67-

positive cells in the indicated number of

animals for each genotype (n = 5 animals).

Results are expressed as mean ± SEM.

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LSL–K-ras G12D;Fap+/+?mice,?consistent?with?previous?reports?(34,?36).?In?contrast,?LSL–K-ras G12D;Fap+/LacZ?mice?exhibited?localized?hyper-plasia?(10?of?10?mice)?and?some?adenomas?(6?of?10?mice),?while?LSL–K-ras G12D;Fap LacZ/LacZ?showed?only?small?lesions?of?focal?hyper-plasia?with?incidental?adenomas?identified?in?only?4?of?11?mice?in?this?group?(Figure?1A).?Overall,?the?tumor/lung?volume?ratio?was?significantly?decreased?from?20.4%?in?LSL–K-ras G12D;Fap+/+?mice?to?7.9%?(P?=?0.02)?in?LSL–K-ras G12D;Fap LacZ/LacZ?mice?(Figure?1B,?left).?Although,?tumor?burden?in?FAP?heterozygous?mice?was?not?statis-tically?different?compared?to?Fap+/+?mice,?there?was?a?trend?toward?reduced?tumorigenesis?(Figure?1B,?left).?Analysis?of?mortality?rates?indicated?a?significant?delay?in?tumor-associated?mortality?in?LSL–K-ras G12D;Fap LacZ/LacZ?compared?with?LSL–K-ras G12D;Fap+/+?mice.?Specifically,?median?survival?increased?from?195?days?for?LSL–K-ras G12D;Fap+/+?mice?to?233?days?and?333?days?for?LSL–K-ras G12D;Fap+/LacZ?and?LSL–K-ras G12D;Fap LacZ/LacZ?mice,?respectively.?As?shown?in?Sup-plemental?Figure?1D,?5?of?11?(~45%)?LSL–K-ras G12D;Fap+/+?mice?succumbed?by?191?days?after?Ad-Cre?instillation?compared?with?3?of?16?(~19%)?of?LSL–K-ras G12D;Fap LacZ/LacZ?mice.?Furthermore,?all?LSL–K-ras G12D;Fap+/+?mice?succumbed?by?day?249,?while?10?of?16?LSL–K-ras G12D;Fap LacZ/LacZ?mice?(63%)?were?still?alive?at?day?300?(Sup-plemental?Figure?1D),?although?all?eventually?succumbed?by?day?410.?Importantly,?the?reduced?tumor?growth?was?associated?with?a?reduction?in?the?proliferative?index?of?tumors?based?on?staining?with?Ki67?(Figure?1A,?bottom,?and?Figure?1C,?right),?while?the?fre-quencies?of?apoptotic?cells?observed?were?comparable?in?tumors?in?the?control?and?FAP-deficient?mice?(data?not?shown).

To?compare?the?phenotype?of?the?tumor?cells?in?LSL–K-ras G12D; Fap+/+,?LSL–K-ras G12D;Fap+/LacZ,?and?LSL–K-ras G12D;Fap LacZ/LacZ mice,?sec-tions?were?double?stained?for?Clara?cell-specific?protein?(CC10)?and?type?II?pneumocyte?surfactant?protein-C?(SP-C).?CC10?and?SP-C?are?specific?markers?for?bronchoalveolar?epithelium,?respectively?(38).?We?found?that?the?tumors?in?mice?of?all?3?genotypes?were?composed?of?SP-C+,?CC10+,?and?CC10+SP-C+?tumor?cells?(Figure?2,?top).?Moreover,?expression?of?FAP?(by?immunohistochemistry;?Figure?2,?bottom)?and?LacZ?(by?histologic?detection?of?β-galactosi-dase?activity;?data?not?shown)?demonstrated?that?TAFs?(based?on?transcriptional?activity?of?the?FAP?loci)?were?present?in?tumors?at?all?stages.?We?did?not?detect?expression?of?FAP?in?normal?lung?tis-sue?by?immunohistochemistry?(data?not?shown).?Thus,?although,?deletion?of?FAP?resulted?in?reduced?tumor?burden,?as?evidenced?by?the?reduced?tumor/lung?volume?ratio,?it?did?not?appear?to?impact?significantly?the?phenotype?of?the?tumor?cells.

The growth of syngeneic transplanted CT26 tumors is reduced in FAP-deficient mice.?Inhibition?of?tumor?growth?of?the?endogenous?tumors?in?Fap-null?mice?may?be?due?to?reduced?tumor?initiation?or?tumor?progression.?Therefore,?to?investigate?whether?deletion?of?FAP?could?inhibit?tumor?progression?after?tumor?initiation?and?to?test?whether?FAP?promotes?the?growth?of?other?tumor?types,?we?extended?our?studies?to?a?syngeneic?transplanted?mouse?tumor?model.?Importantly,?we?did?not?detect?Fap?mRNA?or?pro-tein?in?cultured?CT26?cells?(Supplemental?Figure?2A?and?data?not?shown).?In?contrast,?significant?levels?of?Fap?mRNA?(Supplemen-tal?Figure?2A)?and?protein?(Supplemental?Figure?2B)?were?detect-ed?in?the?CT26?transplanted?tumors.?Consistent?with?previous?reports?in?xenograft?models?as?well?as?primary?human?epithelial?carcinomas?such?as?colon?(15,?39,?40),?breast?(16),?and?pancreas?(12),?these?data,?taken?together?with?the?pattern?of?FAP?expres-sion?in?tumor?sections?(Supplemental?Figure?2B,?left),?indicated?that?endogenous?FAP?is?induced?specifically?on?host-derived?TAFs?and?pericytes?but?not?expressed?in?the?transplanted?tumor?cells?themselves.?We?then?compared?the?growth?of?CT26?colon?cancer?cells?injected?s.c.?into?immune?competent?Fap+/+?and?Fap LacZ/LacZ?BALB/c?mice.?CT26?tumor?growth?was?markedly?reduced?in?Fap-null?mice?(Figure?3A),?similar?to?our?results?in?the?endogenous?lung?tumor?model.?As?shown?above?in?the?endogenous?model,?the?decrease?in?CT26?tumor?growth?in?FAP-deficient?mice?was?associated?with?a?decrease?in?tumor?cell?proliferation?(Figure?3,?B?and?C),?while?the?incidence?of?apoptotic?cells?was?not?affected?(Supplemental?Figure?3,?A?and?C).

Pharmacologic inhibition of FAP protease activity inhibits tumorigen-esis.?We?next?investigated?the?impact?of?inhibiting?FAP?enzymatic?activity?in?both?the?endogenous?lung?tumor?model?and?trans-planted?CT26?tumors?using?a?pharmacologic?approach.?LSL– K-ras G12D;Fap+/+?mice?were?administered?Ad-Cre?as?described?above.?Four?weeks?after?infection?with?Ad-Cre,?mice?were?randomly?sort-ed?into?3?groups?and?treated?with?PT630?(GluBoroPro?dipeptide,?known?to?inhibit?FAP?and?the?closely?related?DPPIV;?ref.?41;?pat-ent?application?publication?no.?US?2007/0072830?A;?Supplemen-tal?Figure?2D),?LAF237?(Vildagliptin,?a?DPPIV?inhibitor;?refs.?42,?43)?or?saline?(vehicle?control)?for?an?additional?4?weeks.?PT630?inhibits?FAP?and?DPPIV?with?K i?in?the?nanomolar?range?(FAP?with?

Figure 2

Immunophenotype of tumors in LSL–K-ras G12D;

Fap mice. Immunofluorescence of CC10

(Clara cells, green; examples indicated by

open arrows), SP-C (alveolar type II cells,

red; examples indicated by filled arrows),

and CC10/SP-C double-positive cells (yel-

low; examples indicated by arrowheads) (top

row). FAP immunostaining of K-ras G12D–driv-

en lung tumors (bottom row). Original mag-

nification, ×20 (top row); ×40 (bottom row).

Scale bars: 100 μ

m.

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IC 50?of?23?nM?and?K i of?5?nM?and?DPPIV?with?an?IC 50?of?3?nM;?ref.?41),?while?LAF237?(Vildagliptin)?is?a?potent?and?selective?competi-tive?inhibitor?of?DPPIV?(K m ,?1.4?×?105?M –1s –1;?K i ,?17?nM;?IC 50,?4–8?nM;?refs.?42,?43).?LAF237?inhibits?DPPIV?at?micromolar?concen-trations?for?DPPIV?but?does?not?inhibit?FAP,?DPPII,?prolyl?oligo-peptidase,?or?aminopeptidase?(42).?Therefore,?LAF237?was?used?to?discriminate?between?the?effects?of?FAP?and?DPPIV.?Interestingly,?LAF237?and?PT630?both?efficiently?reduced?the?lung?tumor?bur-den?compared?with?the?vehicle?treated?control?group?(Figure?4,?A?and?B).?Although?in?another?independent?experiment,?tumor?bur-den?was?again?reduced?in?LSL–K-ras G12D ;Fap LacZ/LacZ mice?compared?with?LSL–K-ras G12D ;Fap +/+?mice?(P

Although?the?genetic?data?indicated?that?deletion?of?FAP?was?sufficient?to?attenuate?tumor?growth,?the?results?from?these?pharmacologic?studies?indicate?that?selective?inhibition?of?DPPIV?is?also?sufficient?to?inhibit?tumor?growth?in?this?model.?Therefore,?we?investigated?the?expression?profile?of?DPPIV?in?the?lungs?prior?to?and?after?administration?of?Ad-Cre.?Interestingly,?immunohistochemical?analysis?established?that?DPPIV?is?indeed?expressed?in?control?lungs,?both?lungs?from?wild-type?mice?treated?with?Ad-Cre?and?uninfected?LSL–K-ras G12D mice?(data?not?shown)?as?well?as?in?lungs?from?mice?bearing?K-ras G12D –driven?tumors?

(Supplemental?Figure?5);?importantly,?in?the?latter?case?the?stain-ing?was?associated?with?nontumor?cells.?Thus,?in?contrast?to?FAP,?DPPIV?is?constitutively?expressed?in?lung,?rather?than?induced?in?response?to?tumor,?but?can?nonetheless?promote?tumor?growth.We?also?determined?the?effect?of?PT630?and?LAF237?on?the?growth?of?CT26?tumors?in?BALB/c?mice.?Two?days?after?CT26?tumor?cells?were?injected?s.c.,?the?mice?were?randomly?divided?into?3?groups?and?treated?with?saline,?PT630,?or?LAF237.?PT630,?but?not?LAF237,?significantly?reduced?CT26?tumor?growth?compared?with?vehicle?control?(P

Similar?to?the?effect?of?genetic?deletion?of?FAP,?PT630?treatment?in?both?tumor?models?and?LAF237?treatment?in?the?endogenous?lung?tumor?model?inhibited?proliferation?(Figure?5).?The?proliferation?indices?of?the?CT26?tumors?were?determined?based?on?Ki67?stain-ing?of?sections?of?tumors?from?the?3?groups?of?CT26?tumor-bearing?

mice?harvested?at?different?time?points?(vehicle?control,?days?10–12;?

Figure 3

CT26 tumor growth and tumor cell proliferation are inhibited in FAP-deficient mice. (A ) CT26 tumor cells were injected s.c. in Fap +/+ and FAP-null BALB/c mice. Tumor size was measured using calipers (n = 14–15 animals per genotype in 2 independent experiments). **P < 0.001; ***P < 0.0001. (B ) Ki67 immunohistochemistry and (C ) proliferative index of CT26 tumors grown in Fap +/+ and Fap LacZ/LacZ mice (n = 5 animals per genotype). Original magnification, ×60. Scale bar: 50 μm. P < 0.0001. Results are expressed as mean ± SEM.

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LAF237,?days?11–14;?and?PT630,?days?16–20)?in?order?to?analyze?tumors?of?approximately?the?same?size?(Figure?5,?A?and?C).?Prolif-eration?was?significantly?reduced?in?tumor?sections?from?animals?treated?with?PT630?(P?

Based?on?our?evidence?that?the?antiproliferative?effect?of?PT630?was?indirect,?we?next?compared?the?morphology?of?comparably?sized?(40–50?mm2)?CT26?tumors?from?the?3?groups?of?mice.?In?tumors?from?vehicle?control–treated?mice,?CT26?tumor?cells?appeared?to?be?spindle?shaped,?and?the?tumors?to?be?appeared?highly?organized?(Figure?5A,?bottom).?Although?tumors?from?mice?treated?with?LAF237?appeared?to?be?somewhat?less?organized?than?those?from?the?saline-treated?mice,?the?greatest?impact?on?tumor?morphology?was?observed?in?the?tumors?from?PT630-treated?mice,?which?were?highly?disorganized?and?less?densely?populated?by?tumor?cells?(Figure?5A,?bottom).

Our?morphologic?data?and?prior?evidence?that?FAP?may?have?the?potential?to?degrade?collagen?(27,?28),?led?us?to?hypothesize?that?deletion?of?FAP?and?inhibition?of?FAP?activity?may?affect?tumor?growth?by?causing?changes?in?the?composition?or?organization?of?the?ECM,?leading?to?dysregulation?of?integrin-mediated?signaling.?Indeed,?we?found?that?tumors?from?Fap-null?mice?compared?with?those?from?Fap?wild-type?mice?and?tumors?from?mice?treated?with?PT630?but?not?with?LAF237?compared?with?those?from?vehicle?Figure 4

Effect of inhibition of FAP and/or DPPIV on tumor

growth. (A) Representative H&E-stained sections of

lung from LSL–K-ras G12D mice treated with vehicle,

LAF237, or PT630 (top row). Higher-magnification

views of regions indicated by asterisks are shown below

(bottom row). Original magnification, ×4 (top row); ×40

(bottom row). Scale bar: 100 μm. (B) Treatment with

PT630 and LAF237 reduced formation of K-ras G12D–

driven lung tumors (n = 5 animals per group). Results

are expressed as mean ± SEM. (C) PT630 treatment

inhibited CT26 tumor growth. Mice were treated by oral

gavage with vehicle control, LAF237, or PT630 twice

daily, starting on day 2, after tumor cell inoculation.

Data represent mean ± SEM (n = 11 animals per group

in 2 independent experiments). ***P < 0.0001 ver-

sus vehicle control. (D and E) Treatment of mice with

PT630 inhibited tumor-associated FAP enzymatic activ-

ity measured ex vivo (D), but not protein levels (E), as

shown by immunoblotting (top panel) and correspond-

ing densitometry (bottom panel). Results are expressed

as mean ± SEM (n

= 10). IOD, image optical density.

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control–treated?mice?showed?an?increase?in?phospho-FAK Tyr397?and?phospho-ERK?(p44/42)?(Figure?6?and?Supplemental?Figure?7).?Although?phosphorylation?of?MAPK?usually?correlates?with?cell?proliferation,?it?can?also?increase?the?expression?of?the?cell?cycle?inhibitor?p21WAF1?protein,?causing?cell?cycle?arrest?(44,?45).?We?found?that?p21WAF1?was?indeed?increased?in?FAP-null?and?PT630-treated?tumors?(P?=?0.04;?Figure?6?and?Supplemental?Figure?7).?These?data?suggested?that?FAP?may?regulate?cell?proliferation,?at?least?in?part,?via?ECM/integrin-mediated?signaling.?These?effects?of?FAP?depletion?and?inhibition?were?fully?recapitulated?in?the?endogenous?lung?tumor?model?(Supplemental?Figure?8).

FAP regulates tumor stromagenesis and angiogenesis.?As?deficiency?in?FAP?expression?or?activity?inhibited?tumor?cell?growth?indi-rectly?and?altered?integrin-mediated?signaling,?we?investigated?its?role?in?stromagenesis?and,?in?particular,?the?content?of?myo-fibroblasts,?which?are?an?important?source?of?ECM?components?and?required?for?angiogenesis.?We?used?immunohistochemistry?to?compare?the?density?of?myofibroblasts,?which?were?identi-fied?using?the?conventional?criteria?of?αSma-expressing?cells?that?were?not in?physical?proximity?to?CD34+?endothelial?cells?(46).?We?found?that?myofibroblasts?were?markedly?less?prevalent (5?fold)?in?tumors?from?PT630-treated?mice?than?in?tumors?from?control?mice?(Figure?7,?A?and?B).?We?therefore?conclude?that?FAP?enzymatic?activity?regulates?recruitment,?proliferation,?survival,?or?differentiation?of?myofibroblasts.

As?angiogenesis,?a?key?event?in?tumor?progression,?is?dependent?on?ECM?remodeling,?proteases,?and?on?the?cell?types?that?express?FAP,?fibroblasts,?and?pericytes?(47,?48),?we?tested?whether?PT630?had?an?effect?on?CT26?tumor?vascularization.?Quantification?of?CD34+?vessels?demonstrated?that?treatment?with?PT630?resulted?in?a?3-fold?decrease?in?tumor?vascularization?compared?with?tumors?from?vehi-cle?control–?and?LAF237-treated?mice?(Figure?7,?A?and?B).?These?data?indicate?that?FAP?plays?an?important?role?in?angiogenesis.

FAP activity regulates stromal collagen in vivo.?The?presence?of?col-lagen?structures?radially?aligned?with?tumor?cells?has?been?sug-gested?to?promote?tumor?progression?and?invasion?(49).?Given?the?deficit?in?myofibroblasts?we?observed?in?tumors?from?mice?treated?with?PT630?and,?on?the?other?hand,?the?reported?in?vitro?collagenase?activity?of?FAP,?we?investigated?the?net?impact?of?inhibiting?FAP?on?collagen?organization?and?content?in?CT26?

Figure 5

PT630 attenuates tumor cell proliferation and

alters tumor morphology. (A and B) Sections of

CT26 (A, top row) and endogenous lung tumors

(B) isolated from mice, treated with either vehi-

cle, LAF237, or PT630, were stained with Ki67

and H&E. The morphology of CT26 tumors from

the same groups of CT26 tumor-bearing mice

was assessed based on H&E staining (A, bottom

row). Solid lines in B show representative tumor

regions used to quantify cell proliferation in each

group. Original magnification, ×40. Scale bars:

100 μm. (C and D) Proliferative indices were

calculated as percentage of Ki67-positive cells

in CT26 (C; n = 10 per group) and endogenous

lung tumors (D; n = 5 per group), respectively.

Results are expressed as mean ± SEM.

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tumors.?CT26?tumors?harvested?at?similar?size?(40–50?mm2)?from?animals?treated?with?saline,?LAF237,?or?PT630?were?stained?with?Picro-Sirius?red,?and?collagen?content?of?total?tumor?extracts?was?assessed?by?hydroxyproline?assay.?Picro-Sirius?red,?an?elongated?dye?molecule,?reacts?with?collagen?and?enhances?its?normal?bire-fringence?due?to?the?fact?that?many?dye?molecules?are?aligned?parallel?to?the?long?axis?of?each?collagen?molecule.?The?collagen?fibers,?in?order?of?decreasing?thickness?corresponding?to?molecu-lar?disorganization,?appear?as?red,?orange,?yellow,?or?green?(50).?When?visualized?under?polarized?light,?collagenous?structures?with?distinct?birefringence?were?observed?in?both?models.?CT26?tumors?in?Fap LacZ/LacZ?mice?exhibited?dramatically?increased?bire-fringence?(orange?and?yellow-green)?and?higher?collagen?content?(~2?fold)?than?those?from?Fap+/+?mice?(Figure?8,?A?and?B).?Simi-larly,?inhibition?of?FAP?activity?by?treatment?with?PT630?resulted?in?an?orange?birefringence,?suggesting?a?decrease?in?the?organiza-tion?of?collagen?when?compared?with?the?extent?of?birefringence?observed?in?tumors?from?LAF237-treated?mice?and,?even?more?so,?when?compared?with?the?extent?of?birefringence?observed?in?the?tumors?from?vehicle?control–treated?mice?(red?birefringence;?Supplemental?Figure?9A).?Furthermore,?quantification?of?total?collagen?as?determined?by?hydroxyproline?content,?demonstrated?that?collagen?content?was?increased?by?62%?(P?

Although?tumor?disorganization?was?not?as?obvious?in?H&E-stained?lung?tumors?as?it?was?in?H&E-stained?CT26?tumors?(Figure?1),?the?impact?of?FAP?on?collagen?content?in?the?lungs?of?endogenous?tumor-bearing?mice?was?comparable?to?that?seen?in?the?CT26?tumors,?based?on?Picro-Sirius?red?birefrin-gence?microscopy?and?quantification?of?hydroxyproline?content.?LSL–K-ras G12D;Fap+/+?mice?predominantly?exhibited?a?red-orange?birefringence,?with?some?green?birefringence,?whereas?tumors?in?LSL–K-ras G12D;Fap LacZ/LacZ?mice?exhibited?a?decrease?in?orange?bire-fringence?and?a?predominance?of?yellow-green?birefringence?(data?not?shown).?Quantification?of?total?collagen?content?revealed?that?although?there?was?no?difference?in?basal?levels?of?collagen?among?the?lungs?of?LSL–K-ras G12D;Fap+/+, LSL–K-ras G12D;Fap+/LacZ,?and?LSL–K-ras G12D;Fap LacZ/LacZ mice?(–Ad-Cre;?Figure?8C),?the?levels?of?collagen?that?accumulated?in?tumor-bearing?lungs?from?Ad-Cre–infected?LSL–K-ras G12D;Fap+/LacZ?and?LSL–K-ras G12D;Fap LacZ/LacZ mice?were?significantly?increased?compared?with?Ad-Cre–infected?

?Figure 6

Deletion of FAP increases p21WAF1 via ECM-mediated

signaling through FAK and ERK. FAK and ERK1/2

were immunoprecipitated from total extracts of CT26

tumors isolated from Fap+/+ and Fap LacZ/LacZ mice, and

the immune complexes were resolved by SDS-PAGE

and immunoblotted for (A) phospho-FAK Y397 and total

FAK and (B) ERK and phospho-ERK. (C) p21WAF1

immunoblot of total CT26 extracts from Fap+/+ and

Fap LacZ/LacZ mice resolved by SDS-PAGE (2 represen-

tative samples from a total of 10 per group are shown

for each immunoprecipitate/immunoblot). Lanes were

run on the same gel but were noncontiguous (white

lines). (D–F) Quantification by densitometry for all 10

samples from each group for each immunoprecipitate/

immunoblot. Results are expressed as mean ± SEM. Figure 7

PT630 inhibits angiogenesis and stromagenesis in CT26 tumors. (A)

Sections of CT26 flank tumors were stained for CD34 (green), αSma

(red), and nuclei (DAPI; blue) and analyzed by epifluorescence micros-

copy. Original magnification, ×60. Scale bar: 50 μm. (B) Blood vessels

indicated by CD34+ endothelial cell and myofibroblasts (αSma+ cells

not in the proximity of CD34+ cells) were quantified from 10 tumors

per group in 2 independent experiments. Results are expressed as

mean ± SEM.

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LSL–K-ras G12D;Fap+/+?mice?(P?

Discussion

Most?epithelial-derived?tumors?are?characterized?by?the?generation?of?mesenchymal-derived?stromal?cells,?including?intratumoral?and?peritumoral?TAFs?and?pericytes?associated?with?the?tumor?vascu-lature.?These?stromal?cells?as?well?as?the?multiple?products?they?release,?such?as?TGF-β,?ECM?components,?and?proteases?(particu-larly?collagen?and?MMPs),?have?been?implicated?in?tumor?growth,?angiogenesis,?invasion,?and?metastasis.?TAFs?are?phenotypically?distinguishable?from?normal?fibroblasts.?Although?their?heteroge-neity?is?yet?to?be?fully?explored,?at?least?a?subset?of?TAFs?have?been?characterized?as?myofibroblasts?based?on?expression?of?αSma.?FAP?has?also?emerged?as?a?marker?of?reactive?fibroblasts?in?tumors?as?well?as?granulation?tissue?and?in?fibrotic?lesions.?Further?studies?are?required?to?fully?reveal?the?relationship?between?the?SMA?and?FAP?expressing?populations?in?tumors,?but?consistent?with?other?recent?reports?(51),?our?unpublished?results?suggest?that?they?are?overlapping?but?not?coincident?populations?both?in?human?and?in?mouse?tumors.?Recent?studies?have?also?established?that?TAFs?are?unique?in?their?capacity?to?markedly?enhance?tumor?growth?compared?with?normal?fibroblasts?(52).?These?properties,?taken?together?with?the?fact?that?the?stromal?cells?are?likely?to?be?more?genetically?stable?than?tumor?cells?and?the?fact?that?they?are?com-mon?across?multiple?tumor?types,?suggest?that?therapies?designed?to?target?the?protumorigenic?mechanisms?mediated?by?stromal?cells,?used?in?combination,?may?enhance?the?efficacy?of?tumor?cell-targeted?therapies.?Thus,?it?is?imperative?that?the?mechanisms?involved?in?stromal?cell–dependent?tumorigenic?processes?be?delineated.?In?this?study,?we?provide?proof?of?principle?that?target-ing?a?cell?surface?protease,?FAP,?expressed?selectively?on?TAFs?and?pericytes?inhibits?tumor?growth?in?diverse?tumor?types?and?that,?at?least?in?lung?tumors,?inhibition?of?the?related?dipeptidyl?pepti-dase,?DPPIV,?also?expressed?on?untransformed?cells,?can?similarly?inhibit?tumor?growth.

Recent?interest?in?FAP?stems?from?its?tightly?regulated?expres-sion,?which?is?restricted?to?pericytes?and?fibroblasts?induced?to?undergo?differentiation?within?the?microenvironment?of?tumors?and?in?pathologic?fibrosis?(for?example,?in?liver?cirrhosis?and?idio-pathic?pulmonary?fibrosis)?(20,?21),?and?the?potential?of?FAP?to?provide?a?common?target?in?multiple?tumor?types.?Two?general?approaches?to?targeting?FAP?have?been?taken?in?preclinical?models?and?in?patients.?In?antibody-based?imaging?studies,?anti-FAP?anti-bodies?exhibited?highly?specific?tumor?targeting?in?patients?(40).?However,?although?a?humanized?anti-FAP?antibody?(mAb?F19;?sibrotuzumab)?was?well?tolerated,?it?showed?no?efficacy?in?a?phase?II trial?for?metastatic?colorectal?cancer?(39,?53).?It?is?important?to?note,?however,?that?this?particular?antibody?does?not?inhibit?FAP?activity?or?have?direct?cytotoxic?activity.?On?the?other?hand,?it?was?

Figure 8

FAP regulates accumulation of col-

lagen in vivo. (A) Sections of CT26

tumors were stained with Picro-Sirius

red and visualized under polarized light

(top row) and bright field (bottom row).

The increase in orange birefringence in

tumors from Fap LacZ/LacZ mice reflects

less organized collagen. Original mag-

nification, ×20. Scale bar: 100 μm.

(B) Collagen content of CT26 tumors

(100 mm2) from Fap+/+ and Fap LacZ/LacZ

mice. Data represent mean ± SEM of

20 tumors per genotype. (C) Collagen

content of lungs from uninfected (n = 3)

mice and LSL–K-ras G12D;Fap+/+ (n = 6),

LSL–K-ras G12D;Fap+/LacZ(n= 6), and

LSL–K-ras G12D;Fap LacZ/LacZ mice (n = 6)

8 weeks after Ad-Cre infection. Results

are expressed as mean ± SEM.

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recently?reported?that?a?monoclonal?anti-FAP?antibody?conjugat-ed?to?maytansinoid,?FAP5-DM1,?induced?long-lasting?inhibition?of?tumor?growth?and?complete?regressions?in?stroma-rich?xeno-graft?models?of?lung,?pancreatic,?and?head?and?neck?cancers?in?immune-deficient?mice,?with?no?evidence?of?toxicity?(11).?The?lat-ter?data?indicate?that?targeting?FAP-expressing?cells?can?effectively?inhibit?tumor?growth?but?do?not?address?the?role?of?FAP?itself?in?promoting?tumor?growth.?Interestingly,?Cheng?and?colleagues?demonstrated?that?antibodies?that?inhibit?the?proteolytic?activity?of?FAP-inhibited?tumor?growth?in?a?xenograft?model,?although?the?mechanism?involved?was?not?determined?(31).

The?importance?of?the?enzymatic?activity?has?been?addressed?more?directly?using?a?second?approach,?employing?inhibitors?of?FAP?enzymatic?activity.?Administration?of?Val-boro-Pro?(PT-100;?Talabostat)?attenuated?tumor?growth?in?a?variety?of?tumor?models?in?mice?(32,?54).?However,?this?particular?compound?inhibits?mul-tiple?intracellular?and?extracellular?dipeptidyl?peptidases?(e.g.,?FAP,?CD26/DPPIV,?DPP7),?so?that?its?effects?could?not?be?directly?attrib-uted?to?FAP.?Finally,?all?the?studies?to?date?have?been?conducted?in?xenograft?models?of?human?tumors?in?immune?incompetent?mice,?and?in?only?one?of?these?studies?was?the?role?of?endogenous?FAP?expressed?on?stromal?cells,?rather?than?on?tumors?cells?forced?to?overexpress?FAP?(31).?In?this?study,?we?interrogated?the?role?of?endogenous?FAP?in?vivo,?by?using?Fap-null?mice?and?by?employing?selective?inhibitors?of?FAP?and/or?DPPIV.?Collectively,?our?results?establish?that?the?enzymatic?activity?of?FAP?itself?promotes?tumor?growth?of?both?endogenous?and?syngeneic?transplanted?tumors?in?immune?competent?mice.?Depletion?of?FAP?expression?and?even?partial?inhibition?of?FAP?enzymatic?activity?was?sufficient?to?indi-rectly?inhibit?tumor?cell?proliferation.?In?addition,?we?demonstrate?that?FAP?plays?a?critical?role?in?tumor?stromagenesis?and?angiogen-esis.?Furthermore,?the?excessive?accumulation?and?disorganization?of?collagen?we?observed?in?FAP-deficient?tumors?indicates?that?FAP?is?an?important?source?of?collagenase?activity?and/or?regulates?other?collagenases?involved?in?modifying?tumor-associated?ECM,?either?directly?or?as?the?result?of?functioning?in?the?recruitment?or?differentiation?of?critical?collagenase-producing?cells.

As?an?endogenous?model,?we?chose?to?study?lung?tumors?in?LSL–K-ras G12D?mice.?The?progression?of?the?tumorigenic?processes?that?develop?in?LSL–K-ras G12D?mice?mimics?that?seen?in?human?lung?adenocarcinoma?(34,?35,?37).?Among?the?different?types?of?lung?cancer?in?humans,?adenocarcinoma?is?the?most?common?type?in?the?US?and?activating?mutations?of?the?K-ras?oncogene?are?found?in?approximately?one-third?of?human?lung?adenocarcinoma?(55).?This?model?also?offers?an?advantage?over?spontaneous?models?in?that?tumor?development?in?this?model?is?controlled?by?the?admin-istration?of?Ad-Cre,?which?allowed?administration?of?the?pharma-cologic?inhibitors?at?a?defined?time?after?induction?of?expression?of?the?oncogenic?K-ras G12D?allele.?Importantly,?we?found?the?devel-opment?of?hyperplasia?and?adenoma?in?this?model?was?associated?with?the?generation?of?tumor-associated?myofibroblasts.?Genetic?deletion?of?FAP?resulted?in?a?marked?reduction?of?K-ras G12D–driven?tumors?but?had?no?impact?on?the?phenotype?of?the?tumor?cells?(Figures?1?and?2).?Despite?the?obvious?advantages?of?the?LSL– K-ras G12D?model,?there?are?also?some?limitations,?including?that,?at?least?until?mice?expressing?a?conditional?FAP?allele?are?devel-oped,?the?stage?of?tumorigenesis?at?which?FAP?is?expressed?cannot?be?controlled.?Importantly,?the?protumorigenic?role?of?FAP?was?also?evident?in?CT26?colon?tumors?and?Panc02?pancreatic?tumors?(data?not?shown)?transplanted?s.c.?into?immune?competent?syn-geneic?hosts.?Our?results?in?these?tumor?models?indicate?that?FAP?is?indeed?an?important?factor?in?multiple?tumor?types?and?dem-onstrate?that,?even?if?FAP?contributes?to?tumor?initiation,?it?also?plays?a?role?in?established?tumors.

An?important?function?of?myofibroblasts?is?the?deposition?of?ECM.?Furthermore,?studies?have?shown?that?collagen?within?tumors?impacts?tumor?development?(56,?57).?More?specifically,?the?presence?of?collagen?structures?radially?aligned?with?tumor?cells?has?been?suggested?to?promote?tumor?progression?and?inva-sion?(49).?Given?the?deficit?in?myofibroblasts?we?observed?in?tumors?from?mice?treated?with?PT630?and?the?important?contri-bution?of?this?cell?type?to?ECM?deposition,?we?expected?we?might?find?that?tumors?from?these?mice?might?also?be?depleted?of?ECM?or?that?the?organization?of?the?ECM?may?be?affected?in?these?tumors.?On?the?other?hand,?in?view?of?the?reported?in?vitro?col-lagenase?activity?of?FAP,?we?thought?we?might?find?that?inhibiting?FAP?activity?could?lead?to?an?accumulation?of?collagen.?Interest-ingly,?we?found?that?the?net?impact?of?inhibiting?or?deleting?FAP?was?to?increase?collagen?accumulation.

The?pharmacologic?studies?presented?clearly?establish?that?the?enzymatic?activity?of?FAP?is?critical?to?promotion?of?tumor?growth?by?FAP?in?the?K-ras–driven?endogenous?lung?tumor?model?as?well?as?in?transplanted?colon?tumors.?Furthermore,?taken?together?with?the?results?of?the?genetic?approach,?the?inhibition?of?tumor?growth?by?PT630?indicates?that?the?inhibition?of?FAP?enzymatic?activity?is?sufficient?to?inhibit?tumor?growth.?Interestingly,?the?results?with?the?DPPIV-specific?inhibitor,?LAF237,?indicate?that?this?closely?related?member?of?the?dipeptidyl?peptidase?family?also?promotes?growth?of?endogenous?lung?tumors?in?LSL–K-ras G12D?mice,?whereas?inhibition?of?DPPIV?alone?had?no?effect?on?the?s.c.?transplanted?colon?tumor?cells.?It?will?be?of?interest?in?future?stud-ies?to?determine?whether?the?contribution?of?DPPIV?to?tumorigen-esis?is?restricted?to?certain?tumor?types?or?whether?this?disparity?reflects?differences?in?the?requirements?for?the?growth?of?endog-enous?and?transplanted?tumors.

Our?data?indicate?that?the?inhibition?of?tumor?growth?in?Fap-null?and?PT630-treated?wild-type?mice?was?due,?at?least?in?part,?to?reduction?in?tumor?cell?proliferation?in?both?of?the?models?that?we?studied.?However,?the?fact?that?PT630?had?no?effect?on?the?growth?of?cultured?CT26?cells?indicates?that?the?effect?in?vivo?was?indirect,?likely?reflecting?the?impact?we?observed?on?the?ECM,?tumor?stroma-genesis,?and/or?angiogenesis,?all?of?which?are?likely?to?be?related. Stromagenesis?is?a?complex?process?that?gives?rise?to?an?intricate?network?of?TAFs,?angiogenesis?(both?blood?and?lymphatic),?and?remodeling?of?the?ECM.?The?TAFs?in?this?“desmoplastic”?stroma?exhibit?an?altered?phenotype,?including?higher?proliferative?index?and,?most?notably,?the?enhanced?production?of?collagens,?hyaluro-nate,?and?epithelial?growth?factors?(2).?Induction?of?FAP?expres-sion?is?a?prominent?feature?of?TAFs?in?the?vast?majority?of?carci-nomas?(13,?16).?FAP?is?also?expressed?on?blood?vessel–associated?pericytes?in?tumors,?and?tumor?vascularization?is?dependent?on?fibroblasts?and?ECM?remodeling,?while?pericytes?are?required?for?vessel?stabilization?(58,?59).?Interestingly,?high?levels?of?VEGF?may?negatively?regulate?pericytes?and?prevent?vessel?maturation.?In?this?regard,?it?will?be?interesting?to?investigate?whether?FAP?or?DPPIV?play?a?role?in?regulating?VEGF,?as?dipeptidyl?peptidases?are?known?to?activate?other?cytokines/growth?factors?(60).?The?data?presented?in?this?study?establish?that?FAP?plays?an?important?role?in?tumor?stromagenesis,?vascularization,?and?ECM?remodeling.?The?effect?on?stromagenesis?may?reflect?a?direct?role?for?FAP?in?the?recruit-

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ment,?proliferation,?survival,?or?differentiation?of?TAFs.?These?pos-sibilities?will?be?investigated?in?future?experiments.?Alternatively,?FAP?may?indirectly?regulate?stromagenesis?through?effects?on?the?tumor?cells?that?induce?stromagenesis?or?through?modification?of?the?ECM.?Indeed,?we?found?an?accumulation?of?disorganized?col-lagen?fibrils?in?tumors?from?Fap-null?mice?and?mice?treated?with?PT630.?TAFs?and?components?of?the?matrix,?including?collagen,?and?more?specifically,?the?presence?of?collagen?structures?radially?aligned?with?tumor?cells?promote?the?growth?of?epithelial?tumor?cells?and?tumor?progression?and?invasion?(1,?49,?56,?57).?Thus,?the?deficiency?in?TAFs?and?altered?ECM?evident?in?the?tumors?from?Fap-null?and?PT630-treated?mice?likely?contribute?to?the?reduc-tion?in?tumor?growth.?This?is?further?evidenced?by?the?increase?we?observed?in?the?CDK?inhibitor?p21?levels?in?tumors?from?Fap-null?and?PT630-treated?mice,?as?p21?is?known?to?regulate?the?ECM-mediated?signaling?pathway?that?activates?cell?cycle?progression?(44).?The?excess?collagen?accumulation,?in?the?face?of?the?reduction?in?SMA-expressing?myofibroblasts?in?these?tumors,?may?appear,?at?first?glance,?contradictory?to?the?fact?that?myofibroblasts?are?believed?to?be?a?major?source?of?collagen.?But?as?discussed?above,?myofibroblasts?may?represent?only?a?subset?of?TAFs?and?other?TAFs?may?also?be?a?major?source?of?collagen.?Furthermore,?other?cells,?possibly?the?tumor?cells?themselves,?may?also?contribute?sig-nificantly?to?matrix?deposition.?However,?as?FAP?has?been?shown?to?have?collagenase?activity?in?vitro,?an?alternative?explanation?for?the?excess?accumulation?of?collagen?may?be?that?FAP?is?an?impor-tant?source?or?regulator?of?collagenase?activity?in?tumors.?In?this?case,?the?loss?of?FAP-dependent?collagenase?activity?may?prevent?the?expected?cleavage?and?turnover?of?collagen?produced?by?the?residual?TAFs.?Another?possibility?is?that?FAP?plays?a?role?in?the?recruitment?or?differentiation?of?critical?collagenase-producing?cells.?Finally,?as?FAP?is?not?expressed?on?endothelial?cells?and?vas-cularization?is?known?to?be?dependent?on?fibroblasts?and?ECM?remodeling?(47,?52),?it?is?likely?that?the?reduction?in?TAFs?and?peri-cytes?and/or?the?alterations?in?the?ECM?also?underlie?the?reduced?vascularization?of?tumors?in?FAP-null?and?PT630-treated?mice.

In?summary,?this?study?provides?proof?of?principle?that?target-ing?FAP?can?inhibit?tumor?growth?in?multiple?tumor?types,?indi-rectly?through?effects?on?stromagenesis,?vascularization,?and?ECM?remodeling.?Human?epithelial-derived?solid?tumors?are?rich?in?FAP-expressing?cells;?for?example,?this?is?the?case?in?colon,?pancre-atic,?and?also?lung?cancer,?which?is?the?leading?cause?of?cancer-relat-ed?deaths?in?both?men?and?women?worldwide?(12,?15,?34).?Thus,?in?addition?to?establishing?the?mechanisms?by?which?a?specific?stromal?cell?surface?protease?promotes?tumorigenesis,?these?stud-ies?indicate?that?further?exploring?tumor?stroma?and,?in?particular,?FAP?as?a?potential?therapeutic?target?in?patients?is?warranted. Methods

Cell culture

CT26.WT,?a?BALB/c-derived?murine?colon?carcinoma?cell?line,?was?obtained?from?ATCC?and?grown?as?monolayer?cultures?in?RPMI?1640?sup-plemented?with?10%?FCS,?10?mM?HEPES,?1?mM?sodium?pyruvate,?2?mM?l-glutamine,?50?μg/ml?gentamycin,?and?20?μg/ml?penicillin-streptomycin.?Cells?were?maintained?in?a?humid?5%?CO2?atmosphere?at?37°C. Animals

Six-?to?eight-week-old?female?BALB/c?and?C57BL/6?mice?were?purchased?from?Charles?River?Laboratories.?LSL–K-ras G12D?mice?(34)?were?provided?by?T.?Jacks?(Massachusetts?Institute?of?Technology,?Cambridge,?Massachu-setts,?USA).?FAP-deficient?mice?(Fap LacZ/LacZ;?ref.?17)?were?obtained?from?W.J.?Rettig?and?A.?Schnapp?(Boehringer?Ingelheim?Pharma?KG,?Ingelheim,?Ger-many)?and?backcrossed?12?generations?to?BALB/c.?LSL–K-ras G12D?mice?were?crossed?with?FAP-deficient?mice?to?generate?Fap LacZ/LacZ,?Fap+/LacZ,?and?litter-mate?Fap+/+LSL–K-ras G12D?mice.?Mice?were?genotyped?as?described?in?Supple-mental?Methods.?All?animal?protocols?used?in?this?study?were?approved?by?The?Wistar?Institute’s?Institutional?Animal?Care?and?Use?Committee,?and?all?the?procedures?were?conducted?according?to?The?Wistar?Institute’s?ethi-cal?committee?guidelines?on?animal?welfare?and?the?Guide for the care and use of laboratory animals?(NIH?publication?no.?85-23.?Revised?1985).

Tumor models

Endogenous lung tumor model.?In?the?LSL–K-ras G12D?model,?lung?adenocar-cinoma?is?initiated?by?Cre-mediated?recombination?via?infection?with?an?adenovirus?expressing?Cre?recombinase?(Ad-Cre;?University?of?Iowa?Gene?Transfer?Vector?Core)?by?intranasal?instillation?as?previously?described?(33–35,?37).?Eight?weeks?after?Ad-Cre?infection,?mice?were?sacrificed?and?lung?tissue?was?harvested?and?analyzed?histologically.?For?drug?treat-ments,?mice?were?randomized?into?3?treatment?groups,?4?weeks?after?infection.?Saline?solution?(vehicle?control),?PT630?(75?μg/dose;?patent?application?publication?no.?US?2007/0072830?A;?PharmaForm?LLC),?or?LAF237?(PharmaForm?LLC)?(100?μg/dose)?was?administered?by?oral?gavage?twice?daily?for?an?additional?4?weeks,?at?which?time?the?lungs?were?harvested,?processed,?and?analyzed?histologically.?The?percentage?of?total?lung?area?occupied?by?tumor?in?each?of?the?5?lobes?was?quantified?by?computer-assisted?morphometry?using?Image-Pro?6.2?on?H&E-stained?sections.?For?modified-“survival”?studies?depicted?in?the?Kaplan-Meier?plots,?LSL–K-ras G12D;Fap+/+?(n?=?11),?LSL–K-ras G12D;Fap+/LacZ?(n?=?17),?and?LSL– K-ras G12D;Fap LacZ/LacZ?(n?=?16)?mice?were?Ad-Cre?infected?and?carefully?moni-tored.?Animals?were?sacrificed?according?to?the?guidelines?set?forth?in?The?Wistar?Institute?guide?for?the?use?of?animals?(>10%?total?body?weight). Syngeneic transplant tumor models.?Six-?to?eight-week-old?female?FAP?wild-type?(Fap+/+)?and?FAP-null?(Fap LacZ/LacZ)?BALB/c?mice?(backcrossed?12?gen-erations)?were?injected?s.c.?with?2?×?105?CT26.WT?cells,?in?the?right?and?left?flanks.?For?drug?treatment,?2?days?after?tumor?cell?inoculation,?mice?were?randomized?into?3?treatment?groups.?Animals?were?treated?by?oral?gavage?(200?μl)?twice?daily?with?saline?solution?(vehicle?control),?100?μg/dose?PT630?(FAP?and?DPPIV?inhibitor),?or?100?μg/dose?LAF237?(Novartis?DPPIV-spe-cific?inhibitor).?Inhibitors?were?provided?by?Point?Therapeutics?Inc.?Tumor?growth?was?monitored?every?2–3?days?by?measuring?2?perpendicular?diam-eters?using?calipers.?Experiments?were?performed?with?7?mice?per?FAP?geno-type?and?in?triplicates?with?5–7?mice?per?drug?treatment?group.

Ex vivo assay of FAP enzymatic activity

FAP?enzymatic?activity?in?tumors?was?determined?as?previously?described?(32),?using?an?immune-capture?assay?with?Z-Gly-Pro-AMC?(MP?Biomedi-cals?LLC)?as?a?substrate.?Ninety-six–well?plates?(Fluoronunc?MaxiSorb)?were?coated?overnight?at?4°C?with?200?μl?of?100?μg/ml?rabbit?polyclonal?anti-FAP?antibody?(provided?by?J.?Cheng,?Fox?Chase?Cancer?Center,?Philadelphia,?Pennsylvania,?USA).?Plates?were?then?washed?with?PBS?and?blocked?with?5%?BSA?for?1?hour?at?room?temperature.?Tumors?were?excised?and?homogenized?in?MPER?(Pierce?Biotechnology?Inc.)?at?10?ml/g?tumor.?Samples?were?centrifuged?at?18,204?g?for?10?minutes.?One?milligram?total?protein?extract?of?tumor?was?added?to?the?coated?wells?(in?a?final?volume?of?200?μl),?incubated?for?1?hour,?and?washed?with?PBS?containing?0.1%?Tween-20.?Reactions?were?carried?out?with?200?μM?Z-GlyProAMC?(FAP,?turnover?number/K m?[k cat/K m],?5.3?×?104–7.4?×?103?M–1s–1;?DPPIV,?k cat/K m,?9–15?M–1s–1)?(7,?24)?for?1?hour?at?room?temperature.?Fluorescence?was?measured?(excitation?and?emission?wavelengths?of?355?nm?and?460?nm,?

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respectively)?using?a?Wallac?1420?Multilabel?Counter?(Perkin?Elmer)?and?normalized?to?a?milligram?of?tumor?weight.?Activity?was?expressed?as?rela-tive?fluorescence?per?milligram?of?tumor?tissue?per?reaction?time. Histology and immunohistochemical analyses

CT26?flank?tumors?were?harvested?and?paraffin?embedded?after?fixation?in?Prefer?(Anatech?LTD)?overnight?at?4°C?followed?by?sucrose?saturation.?Lungs?from?LSL–K-ras G12D?mice?were?inflated?intratracheally?with?10%?normal?buffered?formalin,?fixed?overnight,?and?embedded?in?paraffin.?Four-micron?sections?were?cut?and?stained?with?H&E.?For?immunohistochemistry,?paraf-fin-embedded?sections?were?dewaxed,?and?antigen?retrieval?was?performed?in?10?mM?sodium?citrate?buffer,?pH?7.4,?for?20?minutes?at?95°C.?Slides?were?washed?at?room?temperature?and?hydrated?in?PBS?for?10?minutes.?Endog-enous?peroxidase?activity?was?then?quenched?with?3%?hydrogen?peroxidase?for?10–20?minutes?at?room?temperature.?Sections?were?then?blocked?with?10%?normal?goat?serum?in?PBS?containing?1%?BSA?and?0.1%?Tween-20?for 1?hour.?Endogenous?avidin?and?biotin?were?blocked?using?the?avidin-biotin?kit?(Vector?Labs),?following?the?manufacturer’s?instructions.?Samples?were?then?stained?with?antibodies?against?Ki67?(37?μg/ml;?M7249;?DAKO),?active?caspase-3?(4?μg/ml;?ab2302;?Abcam),?total?ERK1/2?(1:25;?9102;?Cell?Signaling?Technology),?phosphoERK?(1:200;?4370;?Cell?Signaling?Technology),?total?FAK?(1:50;?ab61113;?Abcam)?phospho-FAK Y397?(1:50;?4462G;?Invitrogen),?p21?(20?μg/ml;?ab2961;?Abcam),?FAP?(20?μg/ml;?ab53066;?Abcam),?and?DPPIV?(20?μg/ml;?AF954;?R&D?Systems),?and?isotype-matched?antibodies?were?used?as?negative?controls.?Incubations?were?done?overnight?at?4°C.?Bio-tinylated?secondary?antibodies?were?used?at?4?μg/ml?and?were?detected?with?horseradish?peroxidase,?using?the?Vectastain?Elite?ABC?(Vector?Laboratories)?as?per?manufacturer’s?instructions.?The?proliferative?and?apoptotic?indices?are?expressed?as?the?percentage?of?proliferative?(Ki67-positive)?and?apoptotic?(active?caspase-3–positive)?cells?per?total?cells?in?the?tumor?sections.?For?CT26?tumors,?the?proliferative?indices?were?based?on?30?fields?(objective?magnifica-tion,?×40),?including?10?fields?from?each?of?3?distinct?regions?per?tumor.?Pro-liferation?indices?of?lung?tumors?were?calculated?based?on?10?tumor?nodules?in?each?mouse?(2?from?each?of?5?lobes),?identified?by?morphology. Immunofluorescence

Paraffin-embedded?sections?(4?μm)?were?dewaxed,?followed?by?antigen?retrieval?in?10?mM?sodium?citrate?buffer?for?30?minutes?at?95°C,?with?sec-tions?covered?with?10?mM?sodium?citrate?buffer.?Clara?cells?and?alveolar?type?II?cells?were?stained?as?described?previously?(38),?using?the?polyclonal?antibody?anti-CC10?(4?μg/ml;?sc9772;?Santa?Cruz?Biotechnology?Inc.)?and?the?polyclonal?rabbit?antibody?anti-proSPC?(1:200;?AB3786;?Chemicon),?respectively.?CT26?paraffin-embedded?tumor?sections?were?dewaxed,?fol-lowed?by?antigen?retrieval?as?described?above.?To?block?mouse?IgG,?the?M.O.M.?kit?(Vector?Laboratories)?was?employed.?The?antibody?against?mouse?Sma?(0.4?μg/ml;?clone?1A4;?M0851;?DAKO)?in?M.O.M.?diluent?and?incubated?for?1?hour?at?37°C.?After?3?washes?with?phosphate?buffer?at?pH?7.2?containing?Tween-20?(0.1%)?(5?minutes),?biotinylated?mouse?IgG?was?added?and?incubated?for?30?minutes?at?37°C.?Following?3?washes,?texas?red-streptavidin?(Vector?Laboratories)?was?incubated?for?1?hour?at?37°C.?After?washing,?sections?were?incubated?with?CD34?(4?μg/ml;?clone?MEC14.7;?Abcam)?overnight?at?4°C.?Secondary?antibody?(Alexa?Fluor?488;?Invitrogen)?was?applied?to?sections?for?1?hour?at?37°C.?Nuclei?were?coun-terstained?with?DAPI?for?5?minutes.?SMA+?myofibroblasts?and?pericytes?and?CD34+?blood?vessels?were?quantified?(magnification,?×60)?for?10?ani-mals?per?group?from?2?independent?experiments?as?described?above. Immunoprecipitation and immunoblotting

CT26?tumors?were?dissected?and?lysed?by?homogenization?in?25?mM?HEPES,?pH?7.5,?containing?0.1%?Triton?X-100,?300?mM?NaCl,?0.5?mM?DTT,?0.2?mM?EDTA,?1.5?mM?MgCl2,?containing?20?mM?β-glycerophos-phate,?1?mM?Na3VO4,?10?mM?NaF,?10?mM?sodium?pyrophosphate,?and?protease?inhibitor?cocktail?(Roche)?at?25?mg/ml.?After?incubation?on?ice?for?20?minutes,?samples?were?centrifuged?at?13,000?g.?Equivalent?amounts?(25?μg)?of?protein?(assayed?with?BCA?protein?assay;?Pierce?Biotechnology?Inc.)?were?resolved?by?SDS-PAGE,?transferred?to?PVDF?membranes?(Millipore),?and?probed?with?indicated?antibodies.?Incuba-tion?with?primary?antibodies?(anti-p21?clone?C19?or?anti-FAP;?Santa?Cruz?Biotechnology?Inc.?and?J.?Cheng,?respectively)?and?secondary?anti-bodies?in?5%?milk?in?phosphate?buffer?containing?0.1?%?Tween-20,?with?washes?in?phosphate?buffer?containing?0.1%?Tween-20?between?steps.?For?immunoprecipitation,?equal?amounts?of?protein?(1?mg)?were?pre-cleared?using?anti-rabbit?antibody?for?90?minutes?at?4°C,?followed?by?incubation?with?protein?A-agarose?(Pierce?Biotechnology?Inc.).?Precleared?lysates?were?subjected?to?immunoprecipitation?at?4°C?overnight?with?indicated?antibodies.?Immune?complexes?were?recovered?using?protein?A-agarose.?Products?were?fractionated?as?above?and?blots?were?probed?with?anti-phosphoFAK pY397?or?anti-phosphoERK?antibody.?Primary?and?secondary?antibodies?were?diluted?in?blocking?buffer?(4%?BSA?in?30?mM?Tris-HCl,?pH?7.6,?75?mM?NaCl,?0.05%?Tween-20),?with?washes?in?30?mM?Tris-HCl,?pH?7.6,?75?mM?NaCl,?0.05%?Tween-20.?Blots?were?washed?and?incubated?in?stripping?buffer?(2%?SDS,?62.5?mM?Tris,?pH?6.8,?100?mM β-mercaptoethanol)?at?50°C?for?30?minutes?or?run?in?parallel?and?rep-robed?with?primary?antibodies?against?FAK?or?ERK1/2.?Detection?was?con-ducted?using?ECL?(GE?Healthcare).?Protein?levels?(expressed?as?the?image?optical?density)?were?determined?by?densitometry?using?ImagePro.?ERK?activity?and?active?FAK?were?expressed?as?ratio?of?phosphorylated?form?relative?to?the?total?fraction.

Analyses of collagen content of tumors

Picro-Sirius red staining.?Serial?sections?of?CT26?tumors?were?dewaxed?as?described?above.?Slides?were?incubated?in?picric?acid–saturated?solution?containing?0.01%?Sirius?red?(Sigma-Aldrich)?for?1?hour?in?a?humidified?chamber?at?room?temperature.?After?washing?with?1%?acetic?acid?(15?min-utes)?and?water?(1?minute),?slides?were?alcohol?dehydrated,?mounted,?and?visualized?on?a?Leica?Microscope?under?90-degree?polarized?light. Hydroxyproline assay.?CT26?tumors?(50?mg)?were?cut?and?digested?over-night?at?110°C?in?1?ml?of?6?N?HCl.?After?neutralization?with?6?N?NaCl,?the?pH?was?adjusted?(6.0

Statistics

All?results?are?expressed?as?mean?±?SEM.?Statistical?analysis?was?performed?using?1-way?ANOVA?with?the?Tukey’s?multiple?comparison?test?and 2-tailed?Student’s?t?test?(GraphPad?Prism).?P?values?of?less?than?0.05?were?considered?statistically?significant.

Acknowledgments

Inhibitors?(PT630?and?LAF237?[PharmForm?LLC])?were?provided by?Point?Therapeutics?Inc.?The?authors?acknowledge?W.?Lee?(Uni-versity?of?Pennsylvania)?for?his?technical?assistance?on?the?vascu-

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larization?analysis.?We?also?thank?F.S.?Keeney?and?J.?Hayden?(The?Wistar?Institute?Microscopy?Core)?for?their?assistance?with?the?imaging?data;?R.?Delgiacco?(The?Wistar?Institute?Histotechnology?Core)?for?the?processing?of?tissues;?Denise?DiFrancesco,?Director?of?The?Wistar?Animal?Facility;?and?S.?Berliner?for?help?in?prepar-ing?the?manuscript.?This?work?was?supported?by?NIH?grant?T32?CA09171,?a?Wistar?Cancer?Training?Grant,?a?grant?from?the?Penn-sylvania?Department?of?Health,?and?Point?Therapeutics?Inc.?A.M.?Santos?was?supported?by?the?Irvington?Institute?Fellowship?Pro-gram?of?the?Cancer?Research?Institute.Received?for?publication?February?20,?2009,?and?accepted?in?revised?form?September?28,?2009.

Address?correspondence?to:?Ellen?Puré?or?Angélica?M.?Santos,?The?Wistar?Institute,?3601?Spruce?Street,?Philadelphia,?Pennsylvania?19104,?USA.?Phone:?(215)?898-1570;?Fax:?(215)?898-3937;?E-mail:?pure@https://www.wendangku.net/doc/6a14156006.html,?(E.?Puré);?asantos@https://www.wendangku.net/doc/6a14156006.html,?(A.M.?Santos). Nazneen?Aziz’s?present?address?is:?Vitrimark,?Providence,?Rhode?Island,?USA.

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十款公认最实用的绘图软件评测

十款公认最实用的绘图软件评测 导读： 很多人以为只有专业从事艺术设计类或者画画工作的人才会用到绘图软件，其实不然。在我们的日常生活中，也有许多需要用到绘图软件的职业，工程师、建筑师、原画师、网页设计师、电工师傅等等，在他们的日常工作中都会用到不同类型的绘图软件。 如今市面上有很多种类的绘图软件，但是他们的侧重点都不一样，有的侧重画工业图，有的偏向于艺术设计，还有的偏向于3D画图等，虽然都属于画图软件，但是涉及的方向大不一样。想要正确选择出适合自己的绘图软件，一个个去了解有些不太现实，下面就让小编为大家盘点一下目前公认最好用的10款绘图软件吧！ 一、亿图图示专家：亿图图示Edraw Max是一款跨平台使用的全类型图形图表设计软件，用户可以通过它绘制260多种类型的图表，其中包含常用的：流程图、思维导图、信息图、组织结构图、甘特图、地图、线框图、数据模型图、UML以及网络拓扑图等等。 它拥有丰富的精美模板及矢量符号库，操作简单易上手，能让难以理解的文本和

表格转化为简单清晰的图表。除了具备多种绘图功能，亿图还能与其他办公软件相兼容，可以轻松通过软件将文件导出为Office word、excel、ppt、图片、pdf、html、svg、ps，甚至连Visio格式也不在话下! 二、Visio：是微软公司出品的一款的软件，它有助于 IT 和商务专业人员轻松地可视化、分析和交流复杂信息。丰富的组件库，各种各样的图表支持，和word 的无缝对接是visio的强项，但是由于其价格过于昂贵，并且无法在linux和osx下运行，可以选择的模板也比较少，加上设计风格过于扁平化，所以目前成为一款很容易被国产新秀软件替代的产品。

制图基础C绘图命令快捷键大全

制图基础C绘图命令快 捷键大全 公司标准化编码 [QQX96QT-XQQB89Q8-NQQJ6Q8-MQM9N]

制图基础CAD绘图命令快捷键大全 菜单命令 F1: 获取帮助 F2: 实现作图窗和文本窗口的切换 F3: 控制是否实现对象自动捕捉 F4: 数字化仪控制 F5: 等轴测平面切换 F6: 控制状态行上坐标的显示方式 F7: 栅格显示模式控制 F8: 正交模式控制 F9: 栅格捕捉模式控制 F10: 极轴模式控制 F11: 对象追踪式控制 Ctrl+B: 栅格捕捉模式控制(F9) Ctrl+C: 将选择的对象复制到剪切板上 Ctrl+F: 控制是否实现对象自动捕捉(f3) Ctrl+G: 栅格显示模式控制(F7) Ctrl+J: 重复执行上一步命令 Ctrl+K: 超级链接 Ctrl+N: 新建图形文件 Ctrl+M: 打开选项对话框 AA: 测量区域和周长(area) AL: 对齐(align) AR: 阵列(array) AP: 加载*lsp程系 AV: 打开视图对话框(dsviewer) SE: 打开对相自动捕捉对话框 ST: 打开字体设置对话框(style) SO: 绘制二围面( 2d solid) SP: 拼音的校核(spell) SC: 缩放比例 (scale) SN: 栅格捕捉模式设置(snap) DT: 文本的设置(dtext) DI: 测量两点间的距离 OI：插入外部对相 Ctrl+1: 打开特性对话框 Ctrl+2: 打开图象资源管理器 Ctrl+6: 打开图象数据原子 Ctrl+O: 打开图象文件 Ctrl+P: 打开打印对说框 Ctrl+S: 保存文件 Ctrl+U: 极轴模式控制(F10) Ctrl+v: 粘贴剪贴板上的内容

数学绘图软件有哪些

数学绘图软件有哪些? 导语： 在数学教科书或者教辅书里，需要使用各类数学公式或函数的示意图，这类一般都比较复杂，需要用到专门的软件进行绘制。本文将为你介绍这些常用的数学绘图软件。 免费获取科学插画设计软件：https://www.wendangku.net/doc/6a14156006.html,/science/ 专业的数学教学绘图软件 亿图软件符号库里包含大量数学平面、立体几何需要用到的图形和符号，立体设计、有希腊字母、数字符号、尺寸标注，基本绘图形状、3D框图等等。只需轻轻拖拽就可以快速的调用，不仅可以根据个人喜好、需求调整图形的颜色、大小，还可以自己设计符号并保存到符号库中。软件支持导出PPT、Word、JPG、PDF等十多种格式保存，可在Windows、Linux、Mac多平台操作。

系统要求 Windows 2000, Windows XP, Windows 2003, Windows Vista, Windows 7,Windows 8, Windows 10 Mac OS X 10.10 + Linux Debian, Ubuntu, Fedora, CentOS, OpenSUSE, Mint, Knoppix, RedHat, Gentoo及更多 亿图软件绘制“数学平面、立体几何图”的特点 1.尺寸标尺：拖拽符号库的尺寸标尺，用户可以双击数值根据图形大小修改。 2.支持外部导入：绘制项目管理图时，亿图的软件也支持用户导入外部文档。 3.支持多系统：亿图图示可支持Windows，Mac 和Linux的电脑系统，系统自 动提示用户更新。 4.全能模板：亿图图示会不断更新优质模板，结合用户需求进行设计。

SAI绘图软件快捷键大全

SAI绘图软件快捷键大全、SAI常用快捷键 sai是绘制漫画常用的工具，掌握好快捷键的使用，会让你事倍功半，sai画布旋转快捷键，其实就是ALT+空格然后鼠标左键按住旋转。。。 SAI绘图软件快捷键大全 钢笔工具的↓（我钢笔工具很少用，就这些吧……，搜的） ctrl 在钢笔图层中激活锚点状态，以加锚点的方式调整线条的曲线，也可直接选择锚点进行挑战。 ctrl+拖拉锚点锚点移动。 shift 不加锚点的调整，（注意：按ctrl的是加锚点的调整，除非你点在某个锚点上。） ctrl+shift 复制并移动整个线条的所有锚点。

alt+shift 单纯只移动整个线条的所有锚点。 ctrl+shift 焊接两个锚点，连接线条时非常有用，如两个锚点分别属于两条线条，则自动连接。 alt 删除锚点。 ctrl+alt 笔刷大小调整。 alt+space 旋转画布。 tab 全屏切換 快捷键 space:移动画布 ALT+space:旋转画布 ALT:取色 TAB：全屏显示画布 CTRL+E:向下合并图层（不过我觉得那个向下合并图像的功能比较好用。还会自己帮你开一个图层） CTRL+F：填充 CTRL：移动图层 SHIFT：画直线用的 CTRL+D：取消选区 CTRL：钢笔图层中按住CTRL可以随意移动、增加描点 ALT+CTRL:调整画笔大小

特殊的键操作： 方向键滚动视图 空格+左键拖拽滚动视图 CTRL+左键拖拽移动图层、移动选择部分 CTRL+SHIFT+左键拖拽选择图层并移动 CTRL+空格+左键拖拽视图的变焦框 CTRL+空格+左键单击放大视图 CTRL+空格+右键单击重置视图的放大 CTRL+ALT+空格+左键拖拽视图的变焦框 CTRL+ALT+空格+左键单击缩小视图 CTRL+ALT+空格+右键单击重置视图的缩小 ALT+空格+左键拖拽旋转视图 ALT+空格+右键单击重置视图的旋转 [ 选择小一号的笔刷 ] 选择大一号的笔刷 0～9 选择笔刷浓度 - 切换透明色与前景色 X 切换前景色和背景色 笔刷类工具的键操作： SHIFT+左键拖拽开始将最后描画的位置和拖拽的开始点连接成直线CTRL+ALT+左键拖拽更改笔刷尺寸 ALT+左键单击拾色

绘图软件快捷键大全

绘图软件快捷键大全CAD 3D PS LS Windows Word CorelDRAW 设计吧廊 计算机绘图常用软件快捷键大全 (CAD) (3D) (PS) (LS)(Windows) (Word) (CorelDRAW) CAD常用快捷键 F1: (获取帮助) F2: (实现作图窗和文本窗口的切换) F3: (控制是否实现对象自动捕捉) F4: (数字化仪控制) F5: (等轴测平面切换) F6: (控制状态行上坐标的显示方式) F7: (栅格显示模式控制) F8: (正交模式控制) F9: (栅格捕捉模式控制) F10: (极轴模式控制) F11: (对象追踪式控制) Ctrl B: (栅格捕捉模式控制F9) Ctrl C: (将选择的对象复制到剪切板上) Ctrl F: (控制是否实现对象自动捕捉f3) Ctrl G: (栅格显示模式控制F7) Ctrl J: (重复执行上一步命令)

Ctrl K: (超级链接) Ctrl N: (新建图形文件) Ctrl M: (打开选项对话框) AA: (测量区域和周长area) AL: (对齐align) AR: (阵列array) AP: (加载*lsp程系) AV: (打开视图对话框dsviewer) SE: (打开对相自动捕捉对话框) ST: (打开字体设置对话框style) SO: (绘制二围面2d solid) SP: (拼音的校核spell) SC: (缩放比例 scale) SN: (栅格捕捉模式设置snap) DT: (文本的设置dtext) DI: (测量两点间的距离) OI： (插入外部对相) Ctrl 1: (打开特性对话框) Ctrl 2: (打开图象资源管理器) Ctrl 6: (打开图象数据原子) Ctrl O: (打开图象文件) Ctrl P: (打开打印对说框)

Cad2008绘图快捷键

Cad2008绘图快捷键F1: 获取帮助 F2: 实现作图窗和文本窗口的切换 F3: 控制是否实现对象自动捕捉 F4: 数字化仪控制 F5: 等轴测平面切换 F6: 控制状态行上坐标的显示方式 F7: 栅格显示模式控制 F8: 正交模式控制 F9: 栅格捕捉模式控制 F10: 极轴模式控制 F11: 对象追踪式控制 Ctrl+B: 栅格捕捉模式控制(F9) dra:半径标注 ddi:直径标注 dal:对齐标注 dan:角度标注 Ctrl+C: 将选择的对象复制到剪切板上 Ctrl+F: 控制是否实现对象自动捕捉(f3) Ctrl+G: 栅格显示模式控制(F7) Ctrl+J: 重复执行上一步命令 Ctrl+K: 超级链接 Ctrl+N: 新建图形文件 Ctrl+M: 打开选项对话框 AA: 测量区域和周长(area) AL: 对齐(align) AR: 阵列(array) AP: 加载*lsp程系 AV: 打开视图对话框(dsviewer) SE: 打开对相自动捕捉对话框 ST: 打开字体设置对话框(style) SO: 绘制二围面( 2d solid) SP: 拼音的校核(spell) SC: 缩放比例 (scale) SN: 栅格捕捉模式设置(snap)

DT: 文本的设置(dtext) DI: 测量两点间的距离 OI：插入外部对相 Ctrl+1: 打开特性对话框 Ctrl+2: 打开图象资源管理器 Ctrl+6: 打开图象数据原子 Ctrl+O: 打开图象文件 Ctrl+P: 打开打印对说框 Ctrl+S: 保存文件 Ctrl+U: 极轴模式控制(F10) Ctrl+v: 粘贴剪贴板上的内容 Ctrl+W: 对象追踪式控制(F11) Ctrl+X: 剪切所选择的内容 Ctrl+Y: 重做 Ctrl+Z: 取消前一步的操作 A: 绘圆弧 B: 定义块 C: 画圆 D: 尺寸资源管理器 E: 删除 F: 倒圆角 G: 对相组合 H: 填充 I: 插入 S: 拉伸 T: 文本输入 W: 定义块并保存到硬盘中 L: 直线 M: 移动 X: 炸开 V: 设置当前坐标 U: 恢复上一次操做 O: 偏移 P: 移动 Z: 缩放

常用生物绘图软件下载

常用生物绘图软件下载 导语： 在很多生物教材中的插图大多采用了彩色图片，使教材显得更丰富多彩。作为传播学习信息的一种重要媒介，生物示意图已不再是可有可无的点缀，而是表达学习内容和学习方法的重要部分。那这些生动的示意图是怎么绘制的呢？ 免费获取科学插画设计软件：https://www.wendangku.net/doc/6a14156006.html,/science/ 有什么好用的生物绘图软件？ 好用的生物绘图软件，推荐亿图图示。亿图软件画生物图，可直接使用软件内置丰富的动物细胞和植物细胞相关图形符号，也可以参考软件内的生物模板。若软件内素材没有合适的，也可以使用画笔自己绘制。不用担心的是，亿图软件操作十分简单，相比ps类工具，极易上手。绘制好的图形素材还可以加入素材库，日后可以直接选用。软件支持导出PPT、Word、JPG、PDF等十多种格式保存，可在Windows、Linux、Mac多平台操作。

系统要求 Windows 2000, Windows XP, Windows 2003, Windows Vista, Windows 7,Windows 8, Windows 10 Mac OS X 10.10 + Linux Debian, Ubuntu, Fedora, CentOS, OpenSUSE, Mint, Knoppix, RedHat, Gentoo及更多 亿图软件绘制“生物细胞示意图”的特点 1.时尚的主题：亿图图示为用户提供多样的背景模板，挑选喜欢的模板类型， 让示意图增加趣味性。 2.用户体验：拖拽式操作，自动对齐功能，让你的操作体验更加流畅。 3.云存储服务：绘制完成的模型图，可以保存在云端，再也不担心重要的数据 图表丢失。 4.云存储服务：绘制完成的模型图，可以保存在云端，再也不担心重要的数据 图表丢失。 5.文件恢复：当电脑不小心重启或者死机，软件自带文件恢复功能让您放心绘 图。

autocad画图常用快捷键

CAD快捷键大全常用CAD快捷键汇总 AutoCAD是目前世界各国工程设计人员的首选设计软件，简便易学、精确无误是AutoCAD成功的两个重要原因。AutoCAD提供的命令有很多，绘图时最常用的命令只有其中的百分之二十。 在CAD软件操作中，为使用者方便，于在Windows中工作时一样，利用CAD快捷键代替鼠标。利用键盘快捷键发出命令，完成绘图，修改，保存等操作。这些命令键就是CAD快捷键。 现在就来看看AutoCAD快捷键： 一、CAD快捷键：常用功能键 F1:获取帮助 F2:实现作图窗和文本窗口的切换 F3:控制是否实现对象自动捕捉 F4:数字化仪控制 F5:等轴测平面切换 F6:控制状态行上坐标的显示方式 F7:栅格显示模式控制 F8:正交模式控制 F9:栅格捕捉模式控制 F10:极轴模式控制 F11:对象追踪式控制 二、CAD快捷键：常用CTRL快捷键 Ctrl+B:栅格捕捉模式控制(F9)

dra:半径标注 ddi:直径标注 dal:对齐标注 dan:角度标注 Ctrl+C:将选择的对象复制到剪切板上Ctrl+F:控制是否实现对象自动捕捉(f3) Ctrl+G:栅格显示模式控制(F7) Ctrl+J:重复执行上一步命令 Ctrl+K:超级链接 Ctrl+N:新建图形文件 Ctrl+M:打开选项对话框 Ctrl+1:打开特性对话框 Ctrl+2:打开图象资源管理器 Ctrl+6:打开图象数据原子 Ctrl+O:打开图象文件 Ctrl+P:打开打印对说框 Ctrl+S:保存文件 Ctrl+U:极轴模式控制(F10) Ctrl+v:粘贴剪贴板上的内容 Ctrl+W:对象追踪式控制(F11) Ctrl+X:剪切所选择的内容

常用的建筑制图软件有哪些

常用的建筑制图软件有哪些 导语： 建筑制图软件可以帮助我们将脑中的设计方案付诸实际。常用的绘图软件有哪些呢？你不免心生疑惑。希望你能接着往下阅读，即可解开疑惑。 免费获取建筑平面布置图软件：https://www.wendangku.net/doc/6a14156006.html,/floorplan/ 常用的建筑制图软件有哪些？ 建筑制图软件，或许你听说的最多的是CAD啦、sketch up啦，听起来都很专业，一个外行人想要尝试房屋的平面设计，要用什么软件呢？亿图图示或许可以了解一下！软件操作简单易上手，内置丰富的专业建筑符号及平面布置贴图。设计房屋，从这里开始。

亿图图示软件特色： 1、来自全球超过600万的用户选择下载安装。 2、支持多系统操作：亿图图示可以在Windows，Mac 和 Linux上进行制作。 3、产品升级：亿图软件不断更新升级，重视用户体验度。 4、简单操作：一键式绘制工具帮助用户绘制快捷，方便使用者管理工作项目。 亿图图示建筑平面图绘制符号大全 一. 基本绘图形状

用途：无特别限定意义，可以根据自己的实际用途结合图形形状来选取。 二. 尺寸标注形状 用途：用于标注建筑物的长宽高尺寸。图样除了画出建筑物及其各部分的形状外，还必须准确地、详尽地和清晰地标注尺寸，以确定其大小，作为施工时的依据。图样上的尺寸由尺寸界线、尺寸线、尺寸起止符号和尺寸数字组成。 三. 建筑物核心符号 用途:

指南针：北方是罗盘针通常指向的方向。在平面图中，北方箭头显示了平面图的哪一边是北边。 空调几位：是放置空调的地方。 实心墙：描绘了墙壁的相对厚度 剪式楼梯：通常由楼梯相连的两个主要航程构成，从上方观察时形成“U”形。 扶手：是设计用于抓住手的轨道，以提供稳定性或支撑。 抽水马桶：是厕所的象征。 电梯：是一个移动的楼梯，由一个由马达驱动的无休止的循环带组成，它传达公共建筑物的地板之间的人。 四：电气和电信符号 用途：不同的电气符号用来标明开关，电话线，热水器，水龙头等安装的位置，以及不同地方插座的安装类型（是安装三孔插座，双控插座甚至是四孔插座），以便安装电气时更能方便快捷操作。 五：墙，门，窗户和结构图形

CAD快捷键命令大全图文版文字版键盘版

在CAD操作中我们常用一些快捷键来代替鼠标操作从而提高绘图效率，以下是小编为大家整理的常用快捷键大全，涵盖图文版、文字版、键盘版。 图文版： 文字版： 一、常用功能键 F1: 获取帮助 F2: 实现作图窗和文本窗口的切换 F3: 控制是否实现对象自动捕捉 F4: 数字化仪控制 F5: 等轴测平面切换 F6: 控制状态行上坐标的显示方式 F7: 栅格显示模式控制 F8: 正交模式控制 F9: 栅格捕捉模式控制 F10: 极轴模式控制 F11: 对象追踪模式控制 （用ALT+字母可快速选择命令，这种方法可快捷操作大多数软件。） 二、常用CTRL，ALT快捷键 ALT+TK 如快速选择 ALT+NL 线性标注 ALT+VV4 快速创建四个视口 ALT+MUP 提取轮廓 Ctrl+B: 栅格捕捉模式控制（F9） Ctrl+C: 将选择的对象复制到剪切板上 Ctrl+F: 控制是否实现对象自动捕捉（F3） Ctrl+G: 栅格显示模式控制（F7） Ctrl+J: 重复执行上一步命令 Ctrl+K: 超级链接 Ctrl+N: 新建图形文件 Ctrl+M: 打开选项对话框 Ctrl+O：打开图象文件 Ctrl+P：打开打印对说框 Ctrl+S：保存文件 Ctrl+U：极轴模式控制（F10）

Ctrl+v：粘贴剪贴板上的内容 Ctrl+W：对象追踪式控制（F11）Ctrl+X：剪切所选择的内容 Ctrl+Y：重做 Ctrl+Z：取消前一步的操作 Ctrl+1：打开特性对话框 Ctrl+2：打开图象资源管理器 Ctrl+3：打开工具选项板 Ctrl+6：打开图象数据原子 Ctrl+8或QC：快速计算器 双击中键：显示里面所有的图像三、尺寸标注 DLI:线性标注 DRA：半径标注 DDI：直径标注 DAL：对齐标注 DAN：角度标注 DCO: 连续标注 DCE:圆心标注 LE:引线标注 TOL:公差标注 四、捕捉快捷命令 END：捕捉到端点 MID：捕捉到中点 INT：捕捉到交点 CEN：捕捉到圆心 QUA：捕捉到象限点 TAN：捕捉到切点 PER：捕捉到垂足 NOD：捕捉到节点 NEA：捕捉到最近点 五、基本快捷命令 AA：测量区域和周长（area) ID：指定坐标 LI：指定集体（个体）的坐标AL：对齐（align)

流程图常用的绘图软件

流程图，是一种比较简单的图表，画起来虽然简单，但是却也需要耗费不少时间和精力。说到绘制流程图的工具，可能很多人会想到Office，微软的Word、Excel、PPT确实是办公中使用率最高的软件。但是用来画流程图，并非是最佳的选择。因此，寻找一款能够替代且专业好用的流程图绘制软件，也许是作为流程图用户的您，需要花费大量时间与精力去做的事情。今天，终于不用再去苦苦找寻了。让我来为大家介绍一款超高性价比的流程图软件。 在很多日常用到Linux，Mac系统的人们开始烦恼，似乎就没有一款软件类似Visio，一款软件就能可以解决所有问题。这时，亿图图示出现了。当下受很多人欢迎的绘图软件亿图绘图专家，这款神奇之处在哪里，在这里我给大家介绍一下。 下面是出自设计师们绘制的智能选择颜色模板

绘图小白可以访问亿图软件的动态帮助，点开它，你能找到亿图的产品研发团队准备的软件说明介绍，以及详细的图文、视频教程，让你可以更轻松、更快的熟悉软件，开始绘制你的业务流程图。

不少用户使用亿图绘制一份业务流程图时发现，亿图的功能是符合办公工具在用户心中位置的，可以用来做很多演示要用的图，可以添加很多很难画的图形：

专业的形状是必不可少的，基本流程图形状里具备了所有绘制流程图时需要用的形状： 业务流程图用到的符号很多，能够满足用户这个需求的软件很少。 符号库里的图形是根据模拟真实场景设计的：

这款软件厉害之处是去掉了操作中的“繁文缛节”，简单直接的配合用户画图，但用户依然可以使用工具绘制自己想要的图，最大程度的贴合用户体验。 所有符号的颜色都具备商务、美观、整洁的视觉效果：

AI(Illustrator)常用快捷键大全和技巧

Illustrator常用快捷键大全和技巧 Illustrator的全部快捷键 Illustrator工具箱 移动工具【V】 直接选取工具、组选取工具【A】 钢笔、添加锚点、删除锚点、改变路径角度【P】 添加锚点工具【+】删除锚点工具【-】 文字、区域文字、路径文字、竖向文字、竖向区域文字、竖向路径文字【T】 椭圆、多边形、星形、螺旋形【L】 增加边数、倒角半径及螺旋圈数（在【L】、【M】状态下绘图）【↑】 减少边数、倒角半径及螺旋圈数（在【L】、【M】状态下绘图）【↓】 矩形、圆角矩形工具【M】画笔工具【B】 铅笔、圆滑、抹除工具【N】旋转、转动工具【R】 缩放、拉伸工具【S】镜向、倾斜工具【O】 自由变形工具【E】混合、自动勾边工具【W】 图表工具（七种图表）【J】渐变网点工具【U】 渐变填色工具【G】颜色取样器【I】 油漆桶工具【K】剪刀、餐刀工具【C】 视图平移、页面、尺寸工具【H】放大镜工具【Z】 默认前景色和背景色【D】切换填充和描边【X】 标准屏幕模式、带有菜单栏的全屏模式、全屏模式【F】 切换为颜色填充【<】切换为渐变填充【>】切换为无填充【/】 临时使用抓手工具【空格】 精确进行镜向、旋转等操作选择相应的工具后按【回车】 复制物体在【R】、【O】、【V】等状态下按【Alt】+【拖动】 工具箱(多种工具共用一个快捷键的可同时按【Shift】加此快捷键选取,当按下【CapsLock】键时，可直接用此快捷键切换) Illustrator文件操作 新建图形文件【Ctrl】+【N】 打开已有的图像【Ctrl】+【O】 关闭当前图像【Ctrl】+【W】 保存当前图像【Ctrl】+【S】 另存为... 【Ctrl】+【Shift】+【S】 存储副本【Ctrl】+【Alt】+【S】 页面设置【Ctrl】+【Shift】+【P】 文档设置【Ctrl】+【Alt】+【P】 打印【Ctrl】+【P】 打开“预置”对话框【Ctrl】+【K】 回复到上次存盘之前的状态【F12】 Illustrator编辑操作 还原前面的操作(步数可在预置中) 【Ctrl】+【Z】 重复操作【Ctrl】+【Shift】+【Z】 将选取的内容剪切放到剪贴板【Ctrl】+【X】或【F2】 将选取的内容拷贝放到剪贴板【Ctrl】+【C】

常用地质绘图软件

常用的地质绘图软件 一、地质绘图、矢量化、CAD软件 1. Geomap 3.2地质绘图软件包 版本3.2 平台Windows 98/NT/2000/XP 简介：GeoMap3.2适用于制作各种地质平面图（如构造图、等值线图、沉积相图、地质图等）、剖面图（如地质剖面图、测井曲线图地震剖面图、岩性柱状图、连井剖面图等）、统计图、三角图、地理图、工程平面图（公路分布图、管道布线图等）多种图形。GeoMap地质制图系统能广泛应用于石油勘探与开发、地质、煤炭、林业、农业等领域，也是目前国内在石油地质上应用较广的CAD软件之一。 相关软件还包括以下几个专业制图系统：GeoCon油藏连通图生成系统、GeoCol综合地质柱状图编辑系统、GeoMapD油藏开发制图系统、GeoStra地层对比图编辑系统、GeoMapBank网上图文资料库管理系统、GeoReport地质多媒体汇报系统OE目标评价软件。 2. MAPGIS 版本6.5 平台Windows 98/NT/2000/XP 简介：图形矢量化及编辑软件,是一个大型工具型地理信息系统软件，可对数字、文字、地图遥感图像等多源地学数据进行有效采集、一体化管理、综合空间分析以及可视化表示。可制作具有出版精度的复杂地质图，能进行海量无缝地图数据库管理以及高效的空间分析。具有强大的图形编辑功能。 3. NDS测井曲线矢量化 版本4.16 平台Windows 98/NT/2000

简介：测井曲线矢量化,NDSlog、Ndsmap等 4. SDI CGM Editor 版本2.00.50 平台Windows 简介：CGM绘图工具，包括图形转换及拼图。与Larson CGM Studio相比，有以下优点：1、Larson将已作好的CGM文件，作为整体导入，不能修改; 2、Larson添加的热区不能在同一文件的对象之间跳转。而这些SDI CGM Editor都可以。 5. SDI CGM Office 版本2.00.50 平台Windows 简介：显示CGM v1 - v4, ATA, CGM+, PIP, WebCGM，dwg/dxf, pdf, ps, hpgl, plt, emf, tiff, jpeg,png, bmp & xwd文件。转换CGM文件到CGM, EMF, JPEG, PNG, TIFF & BMP格式。拷贝/粘贴CGM图形到Microsoft Office。 6. SDI Convert 版本7.9.0 平台Windows 简介:可以批量和交互进行各种图形格式之间的相互转化，包括CGM、PS和其它常用光栅文件格式。 7. SDI Dgn 1/9 版本1.12.8 平台Windows

(完整版)CAD制图命令快捷键

CAD制图命令快捷键 L=直线； PL=多段线； U回车=Ctrl+z=后退； D=修改，调整； REC=矩形； C=圆； TR=修剪； O=偏移； XL=放射线； X=分解； CO=复制； M=移动； MI=镜像； EL=椭圆； BR=打断； POL=多边形； LEN=拉长； S=拉伸； ME=等分； E=删除； E回车ALL回车=全部删除； AR=阵列； RO=旋转； SC=比例缩放； END=端点； MID=中点； PER=垂足； INT=交足； CEN=圆心； QUA=象限点；

EXT=拉伸； UCS=三维坐标； ROTATE3D=三维旋转； MIRROR3D=三维镜像； 3A=三维阵列； SURFTA B=曲面网格； TXTEXP=分解文字； CTRL+P=打印； （一）字母类 1、对象特性 ADC, *A DCENT ER（设计中心“Ct rl＋2”）CH, MO *PR OPERT IES(修改特性“Ct rl＋1”) MA, *M ATCH PROP（属性匹配） ST, *S TYLE（文字样式） COL, *COLO R（设置颜色） LA, *L AYER（图层操作） LT, *L INET YPE（线形） LTS, *LTSC ALE（线形比例） LW, *L WEIG HT （线宽） UN, *U NITS（图形单位） ATT, *ATTD EF（属性定义） ATE, *ATTE DIT（编辑属性） BO, *B OUND ARY（边界创建，包括创建闭合多段线和面域） AL, *A LIGN（对齐） EXIT, *QUI T（退出） EXP, *EXPO RT（输出其它格式文件） IMP, *IMPO RT（输入文件） OP,PR *OPT IONS（自定义CAD设置） PRINT, *PL OT（打印） PU, *P URGE（清除垃圾） R, *RE DRAW（重新生成） REN, *RENA ME（重命名） SN, *S NAP（捕捉栅格） DS, *D SETT INGS（设置极轴追踪）

常用的电脑绘图软件有哪些

常用的电脑绘图软件有哪些? 导语： 电脑绘图软件，凡是搜索即可找到许多许多。绘图软件主要可分为国产和国外的，国内的软件中文操作方便，显然会是一种不错的选择。你知道有哪些好用的国产绘图软件呢，一起来认识一下。 常用的电脑绘图软件有哪些？ 电脑绘图软件有办公绘图的有设计绘图的，一般都知道的有PS、CAD等，主要是设计方面的绘图。那么一般办公需要的专业制图有哪些呢？工作中汇报展示需要的图表及流程等，也用PS吗？当然不是了。亿图图示可以了解一下，跨平台绘图软件，工作中的200多种绘图都能搞定！ 立即获取绘图软件：https://www.wendangku.net/doc/6a14156006.html,/download-edrawmax.php 亿图图示软件特色： 1、丰富的模板例子：亿图图示支持超过200种图表绘制，轻松绘完各类图形。 2、专业的图表软件：不仅可以绘制普通图形，还可以绘制组织结构图、思维导图、网络图等。 3、值得信赖的产品：超过六百万次的下载，用户遍布全世界。

4、支持在线分享，生成的网页链接可以在不同的用户终端进行查看。 5、可以使用软件轻松绘制箭头、图框，让办公效率无限提升。 选择亿图图示绘图软件的十大原因 1、国内仅有的，自主研发的全类型图形图表设计软件 亿图图示是一款国产的绘图软件，软件的功能与界面皆由国内工程师独立开发。因此，亿图图示软件在界面设计上符合国人的审美，在功能上也适应国人的操作习惯。在国内，亿图图示是唯一一款全类型图形图表设计软件。 2、绘图类型多样化，一款软件实现多种绘图类型 亿图图示软件可以绘制260种的图形图表，不仅包含常用的思维导图、流程图、信息图、组织机构图、商务图表、户型图；也包含小众的绘图类型，例如网络图、

矢量绘图软件Illustrator快捷键大全

矢量绘图软件Illustrator快捷键大全2005-10-17 15:54作者：yangling出处：Illustrator 责任编辑：Shiny 掌握了快捷键的作用和意义不言而喻，下面我们将矢量绘图软件Illustrator的快捷键分类总结如下。 Illustrator工具箱 移动工具【V】 直接选取工具、组选取工具【A】 钢笔、添加锚点、删除锚点、改变路径角度【P】 添加锚点工具【+】 删除锚点工具【-】 文字、区域文字、路径文字、竖向文字、竖向区域文字、竖向路径文字【T】 椭圆、多边形、星形、螺旋形【L】 增加边数、倒角半径及螺旋圈数（在【L】、【M】状态下绘图）【↑】 减少边数、倒角半径及螺旋圈数（在【L】、【M】状态下绘图）【↓】 矩形、圆角矩形工具【M】 画笔工具【B】 铅笔、圆滑、抹除工具【N】 旋转、转动工具【R】 缩放、拉伸工具【S】 镜向、倾斜工具【O】 自由变形工具【E】 混合、自动勾边工具【W】 图表工具（七种图表）【J】 渐变网点工具【U】 渐变填色工具【G】 颜色取样器【I】 油漆桶工具【K】 剪刀、餐刀工具【C】 视图平移、页面、尺寸工具【H】 放大镜工具【Z】 默认前景色和背景色【D】 切换填充和描边【X】 标准屏幕模式、带有菜单栏的全屏模式、全屏模式【F】 切换为颜色填充【<】 切换为渐变填充【>】 切换为无填充【/】 临时使用抓手工具【空格】 精确进行镜向、旋转等操作选择相应的工具后按【回车】

复制物体在【R】、【O】、【V】等状态下按【Alt】+【拖动】｝ 另:工具箱(多种工具共用一个快捷键的可同时按【Shift】加此快捷键选取,当按下【CapsLock】键时，可直接用此快捷键切换) Illustrator文件操作 新建图形文件【Ctrl】+【N】 打开已有的图像【Ctrl】+【O】 关闭当前图像【Ctrl】+【W】 保存当前图像【Ctrl】+【S】 另存为... 【Ctrl】+【Shift】+【S】 存储副本【Ctrl】+【Alt】+【S】 页面设置【Ctrl】+【Shift】+【P】 文档设置【Ctrl】+【Alt】+【P】 打印【Ctrl】+【P】 打开“预置”对话框【Ctrl】+【K】 回复到上次存盘之前的状态【F12】 Illustrator编辑操作 还原前面的操作(步数可在预置中) 【Ctrl】+【Z】 重复操作【Ctrl】+【Shift】+【Z】 将选取的内容剪切放到剪贴板【Ctrl】+【X】或【F2】 将选取的内容拷贝放到剪贴板【Ctrl】+【C】 将剪贴板的内容粘到当前图形中【Ctrl】+【V】或【F4】 将剪贴板的内容粘到最前面【Ctrl】+【F】 将剪贴板的内容粘到最后面【Ctrl】+【B】 删除所选对象【DEL】 选取全部对象【Ctrl】+【A】 取消选择【Ctrl】+【Shift】+【A】 再次转换【Ctrl】+【D】 发送到最前面【Ctrl】+【Shift】+【]】 向前发送【Ctrl】+【]】 发送到最后面【Ctrl】+【Shift】+【[】 向后发送【Ctrl】+【[】 群组所选物体【Ctrl】+【G】 取消所选物体的群组【Ctrl】+【Shift】+【G】 锁定所选的物体【Ctrl】+【2】 锁定没有选择的物体【Ctrl】+【Alt】+【Shift】+【2】 全部解除锁定【Ctrl】+【Alt】+【2】 隐藏所选物体【Ctrl】+【3】 隐藏没有选择的物体【Ctrl】+【Alt】+【Shift】+【3】 显示所有已隐藏的物体【Ctrl】+【Alt】+【3】 联接断开的路径【Ctrl】+【J】 对齐路径点【Ctrl】+【Alt】+【J】 调合两个物体【Ctrl】+【Alt】+【B】 取消调合【Ctrl】+【Alt】+【Shift】+【B】

常用的绘图软件介绍

常用绘图软件 如果有一定绘图基础就好了，学习更容易。 1、学习时间与内容： （1）CAD：1~7天，看视频，学习3D、2D等。 （2）Solidworks：6个月，看视频与电子档资料，学习3D、2D、防真等。 （3）CATIA：~2个月，看视频与电子档资料，学习3D、防真。 （4）Pro/E：~1个月，看视频与电子档资料，学习3D、防真。 （5）UG：~3个月，看视频与电子档资料，学习3D、防真。 2、部分常用软件介绍： （1）CAD：VersaCAD公司设计，最擅长的是2D设计蓝图制图，多使用“平移”、“镜像”、“放大或缩小”......等命令，很准确，常常用于平面审阅图或加工图。当然，也有立体绘图，不过不是太方便。 （2）Solidworks：法国达索公司的产品之一，最擅长的是3D制图，可以实体建模，比较方便，入门容易，提高难，钣金、防真......等，Motion、Flow simulation、simulation都是很好且实用的功能块，真正做到设计防真一体化。也有2D绘图，但默认页面不是很美观，曲面建模不是太好。成功案例，建过飞机模型，并做相关防真分析。 （3）CATIA：法国达索公司的产品之一，3D建模软件，可以任意建模，比较方便，入门容易，提高也容易，最擅长的是曲面建模，防真分析等高级功能不太好使用。3D模型美观。典型使用于汽车、航空航天、船舶制造、厂房设计、电力与电子、消费品和通用机械制造。（4）Pro/E：美国参数技术公司（PTC)开发的软件。3D建模软件，采用模块方式，进行参数化设计，命令也较多，特征驱动命令不是很丰富，打开文件时需要完整操作，如果对该软件不是很熟悉，可能一个小的、不知道的操作都很难有其他方式替代来解决。 （5）UG：是Siemens PLM Software公司出品的一个产品工程解决方案，它为用户的产品设计及加工过程提供了数字化造型和验证手段，常常用于飞机发动机和大部分汽车发动机设计。

办公绘图软件有哪些

办公绘图软件有哪些 导语： 办公常用的软件，一般是诸如word、ppt和Excel之类的。但有时候需要接受处理一些棘手的任务，就需要使用到绘图软件。如果你是一位绘图新手，不妨使用本文介绍的这款软件，帮助自己顺利绘图。 办公绘图软件有哪些？ 绘图软件还是比较多的，比如做设计用的PS、CAD、AI等，绘制办公需要的图就比较分散了，图种比较多，软件也相应的更多。亿图图示，一款综合性软件，几乎可以绘制所有办公需要的图种。可以画思维导图、流程图、组织架构图、信息图等200多种图形图表，并且每种图表都有相应的模板例子可直接使用。 立即获取绘图软件：https://www.wendangku.net/doc/6a14156006.html,/download-edrawmax.php 亿图图示软件特色： 1、具备齐全的符号，拖入画布即可。 2、搭载智能浮动按钮，可实现一键添加或修改符号。 3、画布智能识别连接点，和强迫症说再见。

4、双击符号，即可输入文本。 5、多套主题样式一键替换，所见即所得。 选择亿图图示的十大理由 1、国内仅有的，自主研发的全类型图形图表设计软件 亿图图示是一款国产的绘图软件，软件的功能与界面皆由国内工程师独立开发。因此，亿图图示软件在界面设计上符合国人的审美，在功能上也适应国人的操作习惯。在国内，亿图图示是唯一一款全类型图形图表设计软件。

2、绘图类型多样化，一款软件实现多种绘图类型 亿图图示软件可以绘制260种的图形图表，不仅包含常用的思维导图、流程图、信息图、组织机构图、商务图表、户型图；也包含小众的绘图类型，例如网络图、UML图、数据库等等。一款软件包含这么多的绘图类型，完全可以绘制工作生活中遇到的各种图表。 3、模板符号丰富，高效省时省力 1000个模板，供使用者直接参考和套用，协助使用者在最短的时间绘制出完整的作品。另外还有20000个矢量符号，可通过鼠标拖拽的方式添加至绘图中。合理地运用模板或符号，就能高效率地绘制出满意的作品。

电脑绘画软件

电脑绘画软件 Painter 或者Corel painter PS 常用的绘图软件有很多，例如： Photoshop可能是现在平常人用的最多的。 Photoshop绘图 Adobe image 简单动画。 coreldraw、freehand功能差不多、说专业排版不能完全说明其作用、我感觉最能表现其功能的是：它们都是矢量图（勾线很好用）。 AutoCAD我用的最多的软件，主要专业制图机械、建筑等施工图纸，二维、三维甚至动画都可以做。 3Dmax建筑、景观、动画都可以做，主要用来建模功能强大到只有想不到没有做不到的。 COOL 3D小软件，主要做装饰字体、动感材质字体，现在好多平面软件都可以做。 Flash、fireworks这两个你常用，就不追叙了。 VRay寄生虫主要渲染3D的，寄生在3Dmax，渲染已做好的贴材质模型。lightscap专业渲染软件，操作有点复杂，不实用，3D完全可以代替，主要给不专业的人用的。 maya功能最强大的动画软件，值得研究，命令好多、功能好多、很惊人。 按功能选绘图软件 Painter优秀的仿自然绘画软件,拥有全面和逼真的仿自然画笔。 SAIEasy Paint Tool SAI，相当小巧，仅3MB左右，功能较少，但操作简单，适用于入门者。 建议 绘电路图有protel，99se，protel dxp 绘机械平面图有Auto CAD 绘平面图Illustrator ，Photoshop，CorelDRAW Photoshop可能是现在平常人用的最多的。Photoshop绘图 Adobe image 简单动画。 coreldraw、freehand功能差不多、说专业排版不能完全说明其作用、我感觉最能表现其功能的是：它们都是矢量图（勾线很好用）。AutoCAD我用的最多的软件，主要专业制图机械、建筑等施工图纸，二维、三维甚至动画都可以做。 3Dmax建筑、景观、动画都可以做，主要用来建模功能强大到只有想不到没有做不到的。 COOL 3D小软件，主要做装饰字体、动感材质字体，现在好多平面软件都可以做。 Flash、fireworks这两个你常用，

(完整版)广联达操作快捷键(大全)

广联达109个快捷键 1 帮助 F1 2 绘图、定义切换 F2 3 按名称选择构件图元 F3 4 左右镜像翻转（点式构件） F3 5 上下镜像翻转（点式构件） Shift+F3 6 改变插入点（点式构件） F4 7 合法性检查 F5 8 三维显示楼层范围 F8 8 汇总计算 F9 9 编辑构件图元钢筋/查看工程量计算式 F11 10 构件图元显示设置 F12 11 选择所有构件图元 Ctrl+A 15 查找图元 Ctrl+F 17 重复 Shift+Ctrl+Z 18 偏移插入点（点式构件） Ctrl+单击 19 输入偏移值Shift+单击 20 报表设计 Ctrl+D 21 上一楼层 + 22 下一楼层 - 23 全屏 Ctrl+5 24 放大 Ctrl+I

25 缩小 Ctrl+U 26 平移-左Ctrl+← 27 平移-右Ctrl+→ 28 平移-上Ctrl+↑ 29 平移-下Ctrl+↓ 30 另存为 ALT+P+A 31 导入图形/钢筋工程 ALT+P+G 32 导入其他工程 ALT+P+I 33 导出GCL工程 ALT+P+T 34 合并其他工程 ALT+P+M 35 打印图形 ALT+P+P 36 删除当前楼层构件 ALT+F+E 37 从其他楼层复制构件 ALT+F+O 38 修改楼层构件名称 ALT+F+G 39 批量修改楼层构件做法 ALT+F+W 40 块复制 ALT+F+Y 41 块移动 ALT+F+V 42 块旋转 ALT+F+R 43 块镜像 ALT+F+M 44 块拉伸 ALT+F+X 45 块删除 ALT+F+D 46 块存盘 ALT+F+S 47 块提取 ALT+F+L 48 构件列表 ALT+E+E

1-3 常用绘图工具及其使用方法

2 讲练结合 1-3常用的绘图工具及其使用方法 2-1 平面图形的画法 多媒体、PPT、作图工具、教材等 1、了解常用的绘图工具及其用法； 2、通过学习与练习，能基本掌握常用绘图工具的使用方法； 3、熟练掌握用三角板、圆规等分线段、圆周和作正多边形等基本作图方法。 三角板、圆规的使用方法；等分圆周和作多边形。 图板和丁字尺及三角板的配合使用 无 学生习题册P6-7

1-3 常用的绘图工具及其使用方法 一、尺规绘图工具和仪器的用法 1、图板和丁字尺------画水平线 2、三角板----------15°的倍数角 3、圆规圆规用来画圆和圆弧。 分规分规用来截取线段、等分直线或圆周，以及从尺上量取尺寸。 4、铅笔：“B”-------软性铅笔，B前面的数字越大，表示铅芯越软（黑）； “H”------硬性铅笔，H前面的数字越大，表示铅芯越硬（淡）。 2-1 平面图形的画法 二、直线段的等分 方法：平行线等分线段成比例 三、等分圆周作正多边形 1、圆的 2、4、8等分：三角板和丁字尺配合 2、圆的 3、6等分-------园的半径

一、复习提问 二、导入新课 三、新课讲授 1、图样的分类 2、投影法的分类及其应用场合 绘图离不开工具，今天要介绍绘图工具及其使用，重点放在使用上。绘图速度的快慢、图画质量的高低，在很大程度上决定于是否能采用正确的绘图方法和按正确的工作程序，自如地运用各种绘图工具绘制几何图形。 1-3 常用的绘图工具及其使用方法 一、尺规绘图工具和仪器的用法 1、图板和丁字尺 2、三角板 3、圆规和分规 （1）圆规圆规用来画圆和圆弧。 （2）分规分规用来截取线段、等分直线或圆周，以及从尺上量取尺寸。 4、铅笔 绘图铅笔用“B”和“H”代表铅芯的软硬程度。“B”表示软性铅笔，B 前面的数字越大，表示铅芯越软（黑）；“H”表示硬性铅笔，H 前面的数字越大，表示铅芯越硬（淡）。 2-1 平面图形的画法