Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice (original) (raw)
Development of endogenous lung tumors is inhibited in FAP-deficient mice. We employed a K-rasG12D–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-rasG12D. Activation of the K-rasG12D allele is achieved by delivery of adenovirus expressing the Cre recombinase (Ad-Cre), resulting in recombination 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 (Supplemental 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-rasG12D;Fap+/+, LSL–K-rasG12D;Fap+/LacZ, and LSL–K-rasG12D;FapLacZ/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 alveolar region (11 of 11 mice), adenomas (11 of 11 mice), and incidental pneumocyte hyperplasia (2 of 11 mice) were already evident in LSL–K-rasG12D;Fap+/+ mice, consistent with previous reports (34, 36). In contrast, LSL–K-rasG12D;Fap+/LacZ mice exhibited localized hyperplasia (10 of 10 mice) and some adenomas (6 of 10 mice), while LSL–K-rasG12D;FapLacZ/LacZ showed only small lesions of focal hyperplasia 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-rasG12D;Fap+/+ mice to 7.9% (P = 0.02) in LSL–K-rasG12D;FapLacZ/LacZ mice (Figure 1B, left). Although, tumor burden in FAP heterozygous mice was not statistically 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-rasG12D;FapLacZ/LacZ compared with LSL–K-rasG12D;Fap+/+ mice. Specifically, median survival increased from 195 days for LSL–K-rasG12D;Fap+/+ mice to 233 days and 333 days for LSL–K-rasG12D;Fap+/LacZ and LSL–K-rasG12D;FapLacZ/LacZ mice, respectively. As shown in Supplemental Figure 1D, 5 of 11 (~45%) LSL–K-rasG12D;Fap+/+ mice succumbed by 191 days after Ad-Cre instillation compared with 3 of 16 (~19%) of LSL–K-rasG12D;FapLacZ/LacZ mice. Furthermore, all LSL–K-rasG12D;Fap+/+ mice succumbed by day 249, while 10 of 16 LSL–K-rasG12D;FapLacZ/LacZ mice (63%) were still alive at day 300 (Supplemental 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 frequencies of apoptotic cells observed were comparable in tumors in the control and FAP-deficient mice (data not shown).
Development of lung tumors in LSL–K-rasG12D;Fap+/+, LSL–K-rasG12D;Fap+/LacZ, and LSL–K-rasG12D;FapLacZ/LacZ mice. (A) Representative 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 display 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-rasG12D;Fap+/+, LSL–K-rasG12D;Fap+/LacZ, and LSL–K-rasG12D;FapLacZ/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.
To compare the phenotype of the tumor cells in LSL–K-rasG12D;Fap+/+, LSL–K-rasG12D;Fap+/LacZ, and LSL–K-rasG12D;FapLacZ/LacZ mice, sections 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-galactosidase 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 tissue 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.
Immunophenotype of tumors in LSL–K-rasG12D;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 (yellow; examples indicated by arrowheads) (top row). FAP immunostaining of K-rasG12D–driven lung tumors (bottom row). Original magnification, ×20 (top row); ×40 (bottom row). Scale bars: 100 μm.
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 protein in cultured CT26 cells (Supplemental Figure 2A and data not shown). In contrast, significant levels of Fap mRNA (Supplemental Figure 2A) and protein (Supplemental Figure 2B) were detected 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 expression 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 FapLacZ/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).
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 FapLacZ/LacZ mice (n = 5 animals per genotype). Original magnification, ×60. Scale bar: 50 μm. P < 0.0001. Results are expressed as mean ± SEM.
Pharmacologic inhibition of FAP protease activity inhibits tumorigenesis. We next investigated the impact of inhibiting FAP enzymatic activity in both the endogenous lung tumor model and transplanted CT26 tumors using a pharmacologic approach. LSL–K-rasG12D;Fap+/+ mice were administered Ad-Cre as described above. Four weeks after infection with Ad-Cre, mice were randomly sorted into 3 groups and treated with PT630 (GluBoroPro dipeptide, known to inhibit FAP and the closely related DPPIV; ref. 41; patent application publication no. US 2007/0072830 A; Supplemental 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 Ki in the nanomolar range (FAP with IC50 of 23 nM and Ki of 5 nM and DPPIV with an IC50 of 3 nM; ref. 41), while LAF237 (Vildagliptin) is a potent and selective competitive inhibitor of DPPIV (Km, 1.4 × 105 M–1s–1; Ki, 17 nM; IC50, 4–8 nM; refs. 42, 43). LAF237 inhibits DPPIV at micromolar concentrations for DPPIV but does not inhibit FAP, DPPII, prolyl oligopeptidase, 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 burden compared with the vehicle treated control group (Figure 4, A and B). Although in another independent experiment, tumor burden was again reduced in LSL–K-rasG12D;FapLacZ/LacZ mice compared with LSL–K-rasG12D;Fap+/+ mice (P < 0.001) as expected, PT630 treatment of the LSL–K-rasG12D;FapLacZ/LacZ mice had no impact on tumor burden when compared with LSL–K-rasG12D;FapLacZ/LacZ mice treated with vehicle control, demonstrating that the effects of PT630 on tumor growth were FAP dependent (Supplemental Figure 4).
Effect of inhibition of FAP and/or DPPIV on tumor growth. (A) Representative H&E-stained sections of lung from LSL–K-rasG12D 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-rasG12D–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 versus vehicle control. (D and E) Treatment of mice with PT630 inhibited tumor-associated FAP enzymatic activity measured ex vivo (D), but not protein levels (E), as shown by immunoblotting (top panel) and corresponding densitometry (bottom panel). Results are expressed as mean ± SEM (n = 10). IOD, image optical density.
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-rasG12D mice (data not shown) as well as in lungs from mice bearing K-rasG12D–driven tumors (Supplemental Figure 5); importantly, in the latter case the staining 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 < 0.0008 and P < 0.0001 at days 14 and 17, respectively; Figure 4C and Supplemental Figure 6). A potent antitumor effect of PT630 was indicated by treatment/control growth ratios of less than 0.40, i.e., 0.37 and 0.27 on days 14 and 17, respectively, which correspond to reductions in tumor size of 63% and 73%. No apparent toxicity was observed in any of the animals treated at the indicated doses of the inhibitors. As was the case in the endogenous lung tumor model, PT630 had no effect on CT26 tumor growth in the FapLacZ/LacZ mice compared with those treated with vehicle control alone (Supplemental Figure 6). Taken together these data indicate that FAP activity, but not DPPIV activity, promotes the growth of CT26 tumors and that the effects of PT630 are indeed mediated by FAP in this tumor model as well.
To determine whether the effect of PT630 on tumor growth was associated with inhibition of FAP enzymatic activity, we compared the tumor-associated FAP activity between the vehicle control– and drug-treated groups. CT26 tumor extracts from PT630-, LAF237-, and vehicle control–treated mice were harvested at different times in order to analyze tumors of similar size (days 10–12, days 11–14, and days 16–20, respectively, corresponding to tumor sizes of between 40 and 50 mm2) and tested for enzymatic activity. FAP activity, determined using Z-Gly-Pro-AMC as substrate, in tumors isolated from animals that were treated with PT630 was decreased 54% compared with those from vehicle control–treated mice (P < 0.05; Figure 4D). The level of FAP activity associated with tumors from animals treated with LAF237 on the other hand was not significantly different from that of the vehicle control group. The decrease in protease activity was due to PT630 reducing FAP activity, as opposed to FAP expression at either the protein (Figure 4E) or mRNA levels (Supplemental Figure 2C and Supplemental Methods), which was comparable in extracts of tumors from all 3 groups of mice. Importantly, these data indicate that the tumor-promoting activity of FAP is mediated via its enzymatic activity and that even incomplete inhibition of FAP enzymatic activity is sufficient to impact tumor growth.
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 staining of sections of tumors from the 3 groups of CT26 tumor-bearing mice harvested at different time points (vehicle control, days 10–12; LAF237, days 11–14; and PT630, days 16–20) in order to analyze tumors of approximately the same size (Figure 5, A and C). Proliferation was significantly reduced in tumor sections from animals treated with PT630 (P < 0.05) compared with tumors from animals treated with vehicle control or LAF237 (P < 0.01; Figure 5C). The incidence of apoptosis as assessed by active caspase-3 immunostaining was quite variable but did not differ significantly in either the tumors from PT630- or the LAF237-treated mice when compared with that observed in tumors from vehicle control–treated mice (Supplemental Figure 3, B and C). Similar results were obtained using a TUNEL assay (data not shown). Importantly, PT630 had no direct effect on CT26 tumor cell viability in vitro as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay (Supplemental Figure 3E), indicating that the decrease in tumor cell proliferation in vivo was due to an indirect effect on the transformed cells. We also assessed the effect of PT630 treatment on cell proliferation and apoptosis in the K-rasG12D–driven lung tumors. PT630 reduced the percentage of Ki67-positive cells (P = 0.006; Figure 5, B and D) with no significant change in apoptosis (data not shown).
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 vehicle, 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.
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 control–treated mice showed an increase in phospho-FAKTyr397 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).
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 FapLacZ/LacZ mice, and the immune complexes were resolved by SDS-PAGE and immunoblotted for (A) phospho-FAKY397 and total FAK and (B) ERK and phospho-ERK. (C) p21WAF1 immunoblot of total CT26 extracts from Fap+/+ and FapLacZ/LacZ mice resolved by SDS-PAGE (2 representative 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.
FAP regulates tumor stromagenesis and angiogenesis. As deficiency in FAP expression or activity inhibited tumor cell growth indirectly and altered integrin-mediated signaling, we investigated its role in stromagenesis and, in particular, the content of myofibroblasts, which are an important source of ECM components and required for angiogenesis. We used immunohistochemistry to compare the density of myofibroblasts, which were identified 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.
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 microscopy. 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.
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 vehicle 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 collagen structures radially aligned with tumor cells has been suggested 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 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 birefringence 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 molecular 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 FapLacZ/LacZ mice exhibited dramatically increased birefringence (orange and yellow-green) and higher collagen content (~2 fold) than those from Fap+/+ mice (Figure 8, A and B). Similarly, inhibition of FAP activity by treatment with PT630 resulted in an orange birefringence, suggesting a decrease in the organization 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 < 0.01) in CT26 tumors from mice treated with PT630 (Supplemental Figure 9B), while LAF237 had no effect on collagen content when compared with tumors from mice treated with vehicle control.
FAP regulates accumulation of collagen 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 FapLacZ/LacZ mice reflects less organized collagen. Original magnification, ×20. Scale bar: 100 μm. (B) Collagen content of CT26 tumors (100 mm2) from Fap+/+ and FapLacZ/LacZ mice. Data represent mean ± SEM of 20 tumors per genotype. (C) Collagen content of lungs from uninfected (n = 3) mice and LSL–K-rasG12D;Fap+/+ (n = 6), LSL–K-rasG12D;Fap+/LacZ (n = 6), and LSL–K-rasG12D;FapLacZ/LacZ mice (n = 6) 8 weeks after Ad-Cre infection. Results are expressed as mean ± SEM.
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 birefringence microscopy and quantification of hydroxyproline content. LSL–K-rasG12D;Fap+/+ mice predominantly exhibited a red-orange birefringence, with some green birefringence, whereas tumors in LSL–K-rasG12D;FapLacZ/LacZ mice exhibited a decrease in orange birefringence 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-rasG12D;Fap+/+, LSL–K-rasG12D;Fap+/LacZ, and LSL–K-rasG12D;FapLacZ/LacZ mice (–Ad-Cre; Figure 8C), the levels of collagen that accumulated in tumor-bearing lungs from Ad-Cre–infected LSL–K-rasG12D;Fap+/LacZ and LSL–K-rasG12D;FapLacZ/LacZ mice were significantly increased compared with Ad-Cre–infected LSL–K-rasG12D;Fap+/+ mice (P < 0.001; Figure 8C). A similar increase in collagen accumulation was observed in tumor-bearing LSL–K-rasG12D;Fap+/+ mice treated with PT630 compared with those treated with vehicle control (P < 0.05; Supplemental Figure 9C). Importantly, PT630 treatment had no effect on the collagen content of tumor-bearing lungs in LSL–K-rasG12D;FapLacZ/LacZ mice (Supplemental Figure 9C). Taken together, these data suggest that FAP endopeptidase activity enhances tumor growth, at least in part, by regulating stromal collagen content, leading to altered integrin-mediated signaling.