Anti-tumor effects of anti-Semaphorin 4D antibody unravel a novel pro-invasive mechanism of vascular targeting agents.

One of the main consequences of inhibition of neovessel growth and vessel pruning produced by angiogenesis inhibitors is increased intratumor hypoxia. Growing evidence indicates that tumor cells escape from this hypoxic environment to better nourished locations, presenting hypoxia as a positive stimulus for invasion. In particular, anti-VEGF/R therapies produce hypoxia-induced invasion and metastasis in a spontaneous mouse model of pancreatic neuroendocrine cancer (PanNET), RIP1-Tag2. Here, a novel vascular targeting agent targeting Semaphorin 4D (Sema4D) demonstrated impaired tumor growth and extended survival in the RIP1-Tag2 model. Surprisingly, although there was no induction of intratumor hypoxia by anti-Sema4D therapy, the increase in local invasion and distant metastases were comparable with the ones produced by VEGFR inhibition. Mechanistically, the antitumor effect was due to an alteration in vascular function by modification of pericyte coverage involving PDGF-B. On the other hand, the aggressive phenotype involved a macrophage-derived Sema4D signaling engagement which induced their recruitment to the tumor invasive fronts and secretion of stromal derived factor 1 (SDF1) that triggered tumor cell invasive behavior via CXCR4. A comprehensive clinical validation of the targets in different stages of PanNETs demonstrated the implication of both Sema4D and CXCR4 in tumor progression. Taken together, we demonstrate beneficial anti-tumor and pro-survival effects of anti-Sema4D antibody but also unravel a novel mechanism of tumor aggressivity. This mechanism implicates recruitment of Sema4D positive macrophages to invasive fronts and their secretion of pro-invasive molecules that ultimately induce local tumor invasion and distant metastasis in PanNETs.


Introduction
One of the main consequences of vessel pruning and inhibition of neovessel growth produced by angiogenesis inhibitors is the increased hypoxia levels produced inside the tumors. Cancer cells can live in hypoxic conditions (1), but growing evidence indicates that tumor cells may escape from this hypoxic environment to better nourished locations, presenting hypoxia as a positive stimulus for invasion (2). In fact, a strong correlation among tumor hypoxia and increased invasion, metastasis and poor patient outcome has been reported (3)(4)(5). In this context, alternative angiogenic targets such as semaphorins are beeing explored (6).
Semaphorins (SEMAs) are a superfamily of secreted or membrane-associated glycoproteins implicated in axonal wiring control, angiogenesis and cancer progression. Semaphorin 4D (Sema4D) is a transmembrane protein of 150 KDa of the IV class of the subfamily of semaphorins involved in the regulation of axon guidance, cell migration in organ development and vascular morphogenesis (7)(8)(9). Three receptors are known for Sema4D: high-affinity receptor Plexin-B1 (PlxnB1), expressed in a wide variety of cell types, intermediate affinity Plexin-B2 (PlxnB2), and low-affinity receptor, CD72, mainly expressed in cells of the immune system (10,11). Sema4D is highly expressed in the membrane of most frequent solid tumors, including breast, prostate and colon (12), and also in tumor associated macrophages (TAMs), with a relevant role in tumor invasion, angiogenesis and metastasis (13). High expression levels of Sema4D have been also reported in tumor stroma (14). Due to proteolytical cleavage by matrix metalloproteinase type 1 (MT1-MMP, also known as MMP14) a Sema4D soluble form is released (15,16), allowing to act through PlxnB1 on endothelial cells and promoting angiogenesis which permits tumors to be nourished with the necessary nutrients and oxygen to continue its growth (12). In fact, there is a completed Phase I clinical trial to evaluate the safety and tolerability of intra-venous (IV) administration of an antibody anti-Sema4D VX15/2503 (Vaccinex Inc, Rochester, NY) in patients with advanced solid tumors (ClinicalTrials.gov identifier: NCT01313065) (17).
In this study, using a spontaneous mouse model of pancreatic neuroendocrine cancer (PanNET), RIP1-Tag2 mice, we describe alteration of tumor vascular function by the use of a vascular-targeting agent anti-Sema4D antibody that consequently impairs tumor growth. Unlike VEGF/R blockade, induction of intratumor hypoxia is not observed after anti-Sema4D therapy, but the increase in local invasion and distant metastases are comparable with anti-VEGFR2's effects. This hypoxia-independent mechanism of increased aggressive phenotype is associated with recruitment of TAMs as mediators of increased invasion and dissemination of tumor cells after anti-Sema4D treatment. Mechanistically, anti-Sema4D antibody induces a Sema4D signaling engagement in the membrane of macrophages not only for their motility and recruitment to the tumor invasive fronts, but also for increased secretion of stromal derived factor 1 (SDF1). In turn, SDF1 enhances tumor cell invasive behavior via CXCR4 and triggers the malignant PanNET phenotype in anti-Sema4D treated RIP1-Tag2 mice. Finally, we also present clinical evidence that support a role for Sema4D and SDF1 overexpression in human macrophages and an association of Sema4D and CXCR4 in PanNETpatients tumor progression.

Animal model and Therapeutic Trials
Transgenic RIP1-Tag2 mice have been previously reported (18). Animal housing, handling and all procedures were approved by our institution's ethical committee and Government committees. Tumor volume, type, invasiveness and hemorrhagic phenotype was determined as previously described (19). Four week-long treatments in RIP1-Tag2 mice started at 12 weeks of age with: 1) Anti-Semaphorin 4D Mab67 function blocking murine IgG1 antibody (anti-Sema4D) kindly provided by Vaccinex Inc, Rochester, NY (20), 2) anti-VEGFR2 blocking antibody (DC101) purified in our laboratory or 3) ChromPure Mouse IgG1 whole molecule as isotype control (Jackson Immuno Research Laboratories, Inc). All dosed at 1mg/animal once a week IP except for anti-VEGFR2 which was administered twice-a-week as previously described (21).

Protein analysis and RNA Analysis
Tumor samples and βTC4 and RAW264.7 cell lysates were analyzed by WB with: c-met  Table 1) and SDF1 and CXCR4, HPRT1 (mouse and human), and cMET and β-ACTIN (mouse) Taqman probes (Applied Biosystems).

Mouse Omics and Clinical data analysis
Gene expression data from different stages of RIP1-Tag2 mice (GEO Omnibus ID -GSE73514) and human mRNA transcriptomes from a core independent clinical gene expression dataset of PanNET (GEO Omnibus ID -GSE73338) patients were used (22,23). For RIP1-Tag2 mice data, primary tumors (n=5) and metastases (n=3) samples were compared. For the human study, normal pancreatic islet samples (n=4), nonfunctional Europe PMC Funders Author Manuscripts samples (n=63), which were termed primary tumors, and their corresponding metastases (n=7) were evaluated. To further study the malignization process, primary tumors were divided into two subcategories, non-malignant and malignant, according to the clinical history of the patients (23).

Statistical Analysis
Results are presented as mean + SD, except for transwell assays, which results are presented as mean + SEM. The statistical tests are noted in each figure and significance follows * p<0.05, ** p<0.005, *** p<0.001, **** p<0.00001 consensus.

Treatment with anti-Sema4D exerts an antitumor and prosurvival effect
Initially, the presence of Sema4D and its high affinity receptor PlxnB1 was evaluated. Sema4D was found to be highly expressed in the membrane of scattered single cells inside tumor parenchyma with a pattern compatible with immune cells and weakly expressed in the membrane of tumor cells (Supplementary Figure 1A), consistent with previous reports (15,17). PlxnB1 was immunodetected in a 30% of vascular structures (Supplementary Figure 1B). To assess the effects of anti-Sema4D treatment, we used a specific antibody (anti-Sema4D, Mab67 Vaccinex) (20) in RIP1-Tag2 mice and focused on tumor growth and expansion phase of islet carcinoma. Therapeutical regimes included 2 or 4 weeks anti-Sema4D treatment along with treatment with DC101, a well described blocking monoclonal antibody of VEGFR2 (21). We could determine that 4 weeks therapy produced an inhibition in tumor growth similar to the one observed after anti-VEGFR2 (α-VR2) treatment ( Figure  1A), which promoted an extension of lifespan of treated mice ( Figure 1B). These results suggest an anti-tumor benefit of anti-Sema4D therapy in terms of tumor shrinkage and lifespan increase in mice.

Altered vessel structure and functionality
A qPCR for angiogenesis related genes such as angiopoietins and platelet-derived growth factor receptors was modified after the treatment (Supplementary Table 1). Suprisingly, in contrast to differences observed after anti-VEGFR2, treatment with anti-Sema4D did not show any differences in number of vessel structures ( Figure Figure 2A). Together, these data indicate that anti-Sema4D treatment does not produce a classical anti-angiogenic effect at the endothelial level on RIP1-Tag2 model.
To mechanistically understand why anti-Sema4D does not exert a direct anti-angiogenic effect, we evaluated the presence of membrane-bound or soluble Sema4D forms in our model. As shown in Supplementary Figure 2B, in RIP-Tag2 tumors we can only detect Sema4D transmembrane full-length form (150 KDa) and not detect any soluble form (110-115 KDa). As the soluble form has been associated to angiogenesis, the lack of this soluble form is consistent with a lack of antiangiogenic effects of anti-Sema4D.
Since many other cellular types such as pericytes play fundamental structural and functional roles in blood vessels, pericyte coverage was evaluated to determine whether they were subjected to a further structural change after anti-Sema4D therapy. Pericytes positive for Desmin and NG2 were increased after anti-Sema4D treatment whereas the number of α-SMA positive pericytes was decreased ( Figure 1G; Supplementary Figure 3A). This alteration in pericyte profile suggests a switch to a more immature vessel type associated to vascular remodeling. In addition, after anti-Sema4D therapy there is a nearly two-fold increase in the number of PlxnB1 positive structures (Supplementary Figure 3B). Next, we checked whether these changes in pericyte coverage occur in PlxnB1 possitive vessels (Supplementary Figure 3C). No difference in pericyte coverage between the PlxnB1 positive or negative endothelial cells was observed, evidencing that pericyte coverage is independent from the expression of PlxnB1 in endothelial cells. This suggests an indirect crosstalk between endothelial cells and pericytes.
Based on previous work (24), where Sema4D treatment of endothelial cells elicits production of PDGF-BB and promotes differentiation of mesenchymal stem cells into pericytes, thus producing pericyte proliferation, chemotaxis, and association with HUVECs in a capillary network, we checked PDGF-BB expression. ELISA assay of PDGF-BB showed a slight decrease in PDGF-BB levels in α-Sema4D treated tumors when compared to control tumors (Supplementary Figure 4). This result was concordant with our RNA analysis in which a decrease in PDGF-BB levels was also observed (Supplementary Table  1).
To assess if altered pericyte coverage after anti-Sema4D had further consequences in vascular functionality, we checked vascular integrity. We evaluated a vascular leakage parameter such as extravassation of erithrocytes (microhemorrhaging) in the form of tumor hemorrhagic phenotype. The percentage of hemorrhagic tumors after 2 weeks of anti-Sema4D therapy was significantly reduced when compared to control animals, although this reduction was even stronger with anti-VEGFR2 treatment ( Figure Figure 5B). CD72 was rather found to be expressed in single cells, suggestive of its expression in cells of the immune system infiltrated in tumor stroma (Supplementary Figure 5C). This result is further confirmed by a double costaining of CD72 and F4/80 positive macrophages (Supplementary Figure 5D).

Increased invasiveness and metastasis after anti-Semaphorin 4D treatment
Anti-Sema4D treatment increases the number of highly invasive tumors progressively, similar to the effect of anti-VEGFR2 (Figure 2A and B). While the majority of control tumors were predominantly encapsulated or microinvasive, treated tumors presented wide fronts of invasion encroaching into adjacent acinar tissue. Significantly, this effect was further exacerbated when anti-Sema4D therapy was maintained for 4 continuous weeks ( Figure 2B). Study of livers and peripancreatic lymph nodes (LN) revealed that anti-Sema4D treated mice more frequently contained enlarged LN containing tumor cells and distant metastasis to the liver ( Figure 2C). The incidence of LN metastasis grew from the 30% in untreated controls to more than 70% in treated groups, indicating that similarly to what happens with VEGFR2 inhibition, anti-Sema4D treatment promotes an increase in LN metastasis ( Figure 2D, left). Incidence of liver metastasis was 2-fold higher in those mice that had received an antiangiogenic treatment, either anti-VEGFR2 or anti-Sema4D ( Figure  2D, right). When combining anti-Sema4D and anti-VEGFR2, results in tumor burden, survival, invasiveness and metastases incidence were identical as in anti-VEGFR2 alone (Supplementary Figure 6). This lack of additional effectof anti-Sema4D evidences the predominant role of VEGF in triggering angiogenesis over Sema4D in this tumor setting.
Overall, the data presented here demonstrate that anti-Sema4D treatment promotes the acquisition of an adaptive resistance, with similar effects of the complete and lasting inhibition of angiogenesis caused by the use of anti-VEGFR2 or TK inhibitors as sunitinib and sorafenib (19,25).

Malignization after anti-Sema4D treatment is not produced by known mechanisms
Treatments targeting tumor vasculature are described to produce an increase in hypoxia as a consequence of the antiangiogenic effect. Surprisingly, anti-Sema4D treatment did not induce hypoxia in short-term treated tumors, as demonstrated by the presence of pimonidazole adducts or by the increase in the expression of hypoxia-response genes such as Glut1 ( Figure 3A) in anti-VEGFR2 treated tumors. Quantification of this event at longer treatment regimes confirmed this observation ( Figure 3B-C). Taken together, these data suggest that a hypoxia independent pathway is responsible for the increase in invasion and malignization.
Up to now, the best described mechanism of tumor aggressiveness after anti-vascular inhibition in RIP1-Tag2 tumors involves hypoxia and c-met activation (26,27). RNA analysis of untreated and anti-Sema4D treated tumors revealed that there were no changes in c-met expression ( Figure 3D). We then assayed the presence of its precursor protein and its active form, phosphorylated c-met, by western blotting, using HGF (c-met natural ligand) to stimulate cells. No expression of the precursor and any activation of c-met signaling pathway was observed, neither in the untreated nor in the anti-Sema4D treated conditions ( Figure 3E). Similarly, even if βTC4 cells express c-met at low transcriptional levels, there is no pathway activation in response to anti-Sema4D or HGF ( Figure 3F-G). Overall, these data suggest that malignization effects in RIP1-Tag2 mice are restricted to an indirect effect of Sema4D over tumor cells, rather than to a direct action of the pro-angiogenic molecule upon tumor cell derived c-met. Moreover, a retrograde effect of Sema4D over tumor cells was discarded since no changes in cell adhesion, de-adhesion or proliferation of RIP1-Tag2derived βTC4 tumor cells were observed (Supplementary Figure 7).

Anti-Sema4D treatment produces an increase in tumor-associated macrophage (TAM) migration
Among all immune cells expressing Sema4D (28-31), a relevant role in pro-tumorigenic processes has been specifically described for lymphocytes and TAMs (13,14). CD3e-positive T-lymphocytes infiltrated in the RIP-Tag2 tumor parenchyma were very scarce and, while anti-Sema4D treatment produced an increase in their numbers, the absolute amount was too low to consider them functionally relevant (Supplementary Figure 8). On the other hand, a co-staining of macrophage marker F4/80 with Sema4D in our tumors showed that most macrophages did not express Sema4D, few expressed it with high intensity and some only in certain areas of the cell ( Figure 4A). However, we found a visible higher amount of Sema4D positive macrophages after anti-Sema4D therapy ( Figure 4A) and the total number of macrophages was significantly increased ( Figure 4B). In fact, while the number of Sema4D negative macrophages was maintained invariable after the therapy ( Figure 4C), the number and percent of Sema4D positive macrophages increased after short term anti-Sema4D treatment ( Figure 4D). In conjuction, these data demonstrated that, in vivo, there was a change in the number and phenotype of TAMs after anti-Sema4D treatment. In order to functionally validate its consequences, the migration properties of a Sema4D-expressing murine macrophage cell line, RAW264.7 (Supplementary Figure 9A), were evaluated after anti-Sema4D treatment. As shown in Figure 4E, there was an increase in migration of RAW264.7 cells after anti-Sema4D therapy, which occurred in a dose dependent manner (Supplementary Figure 9B). Moreover, the addition of exogenous recombinant Sema4D did not reduce basal macrophage migration, indicating the requirement for Sema4D expression in cell membrane for the antibody to have an effect ( Figure 4E). To decipher the underlying mechanism, effective knockdowns of the ligand Sema4D and its two receptors expressed in RAW264.7 cells, CD72 and PlxnB2 were generated (Supplementary Figure 9C). Interestingly, we observed that there was no change in migratory capacity of RAW264.7 cells in any of the gene knockdowns ( Figure 4F). Moreover, anti-Sema4D treatment continued to produce the same increase of migration in all gene silencing conditions except for shSema4D cells ( Figure 4G; Supplementary Figure 9D). Altogether, our results define a receptor-independent and Sema4D-requirement for antibody induction of migration and they demonstrate that Sema4D needs to be expressed in the membrane of the cells for the antibody to have an effect. Thus, all these data define an antibody-induced retrograde signaling engagement of Sema4D which has already been previously published for this family of transmembrane proteins in different settings (reviewed in 32 and 33).
Most macrophage activity is mediated by cytokines and chemokines that act in autocrine fashion and paracrine fashion, upon other macrophages or even upon other cells from the tumor ecosystem. Aiming to delve into macrophage study, we performed a mass spectrometric analysis (LC-MS/MS) of secreted proteins (secretome) composing RAW264.7 conditioned media previously stimulated with anti-Sema4D. The proteomic approach resulted in the identification of more than a thousand proteins (Supplementary Table 2). Using Gene Set Enrichment Analysis (GSEA) bioinformatics tool, we showed a statistical enrichment in proteins related to important macrophage functions: cell migration, cell projection, cytoskeleton and RAC1 pathway (grouped in migration); DNA replication and cell cycle (grouped in proliferation); FCγR mediated phagocytosis and immunological  Figure 10). Taken together, the analysis of the secretome by proteomic profiling suggests a direct effect of Sema4D upon macrophage activity, specially affecting their migration, proliferation and activation.

Tumor-associated macrophages are promoting invasion in βTC4 cells as a response to anti-Sema4D treatment
To check the behaviour of TAMs in tumor periphery, the number of macrophages in the perimeter of the base protrusions of invasive fronts was determined after co-staining Sema4D with F4/80 macrophage marker and the RIP1-Tag2 tumor cell marker insulin ( Figure 5A). Contrary to the intratumoral results, the number of macrophages in the invasive fronts remained unaltered after anti-Sema4D treatment ( Figure 5B). The number of peritumoral Sema4D negative macrophages decreased, while the number of Sema4D positive macrophages and their percentage are strongly increased after treatment ( Figure  5D). The abrupt change in macrophage number and phenotype may indicate a role for these cells in the invasive and malignization process that occurs after the therapy.
To confirm this hypothesis, an in vitro matrigel invasion assay using βTC4 cells was performed. The addition of conditioned medium of RAW264.7 cell line treated with anti-Sema4D significantly increased the invasive properties of βTC4 cells ( Figure 5E). Nevertheless, conditioned media from neither Sema4D, CD72 nor PlxnB2 knockdown RAW264.7 cells did not recapitulate this tumor cell invasion increase ( Figure 5F). On the other hand, conditioned media of anti-Sema4D treated shSema4D RAW264.7 cells did not induce an increase, but rather a decrease of tumor cell invasion (Supplementary Figure 11). Therefore, Sema4D retrograde signaling engagement by anti-Sema4D produces a switch of the macrophage phenotype that potentiates tumor cell invasion in RIP1-Tag2, probably by promoting secretion of a pro-invasive molecule.

SDF1/CXCL12 is responsible for promoting invasion as a response to anti-Sema4D treatment
In pursuance of identifying the pro-invasive molecule secreted by macrophages and responsible for tumor cell invasion after anti-Sema4D therapy, a mouse cytokine array was performed in supernatants of RAW264.7 conditioned media. Even though not many significant changes were observed between different treatment conditions (Supplementary  table 3), a statistically significant increase in stromal cell-derived factor 1 (SDF1, also known as CXCL12) molecule was detected in anti-Sema4D treated supernatant ( Figure 6A). An ELISA analysis of secreted SDF1 revealed an increase in anti-Sema4D condition which was not observed neither in control or treated Sema4D knockdown macrophages ( Figure  6B), nor in cells treated with recombinant PlxnB2 (Supplementary Figure 12A) or receptorknockdown cells (Supplementary Figure 12). We validated SDF1 as a possible macrophage secreted candidate responsible for cancer cell invasion in the RIP1-Tag2 model by an invasion assay in the in vitro setting. As expected, βTC4 cells responded to recombinant SDF1 stimulation in vitro by increasing their invasion ( Figure 6C). This phenomenon was inhibited when CXCR4 receptor was blocked by its antagonist AMD3100. In addition, the increase observed in βTC4 cells' invasion after anti-Sema4D treated conditioned media addition is comparable to the one produced when exogenous SDF1 was added to IgG1 treated conditioned medium ( Figure 6D). Consistently, when AMD3100 was added to anti-Sema4D treated conditioned medium, the invasive capability of βTC4 cells dropped to basal levels, confirming that SDF1 is one of the factors secreted by macrophages after anti-Sema4D treatment responsible for tumor cell invasion.
Finally, we sought to check whether in RIP1-Tag2 model the SDF1/CXCR4 signaling axis was present and affected by anti-Sema4D treatment. We found an increased trend of both CXCR4 and SDF1 RNA expression in treated tumors (Supplementary Figure 12C-D), that was further confirmed by immunohistochemistry ( Figure 6E-F). CXCR4 receptor appears to be expressed homogeneusly by RIP1-Tag2 tumor cells, albeit at low levels in control samples and highly present in anti-Sema4D treated mice ( Figure 6E), possibly due to an SDF1-induced positive feed-forward mechanism (34). Indeed, CXCR4 is naturally present in the tumor progression stages of RIP1-Tag2 mice, showing expression in metastases, both in control and anti-Sema4D treated mice (Supplementary Figure 12E-F). Therefore, anti-Sema4D treatment seems to exacerbate an already existing CXCR4-mediated metastasis mechanism. As expected, SDF1 was found both in cells with a vascular phenotype and also in round shaped cells compatible with immune infiltrates. The count of the latter showed an increase in SDF1 positive round cells after the treatment ( Figure 6F). A costaining of both Sema4D and SDF1 showed an enrichment in SDF1/Sema4D double positive cells after the treatment (Supplementary Figure 12G and Figure 6G). Since endothelial cells also express SDF1, we analyzed the behavior of HUVEC cells in response to anti-Sema4D, observing no changes in gene expression but an increase in SDF1 release after the treatment (Supplementary Figure 13).
Altogether, the in vivo results may suggest a tumor-independent origin of SDF1 that could bind to its receptor in RIP1-Tag2 tumor cells to exert its activity. Furthermore, a deeper analysis associating SDF1 levels and invasive capacity of the tumor front revealed a relationship between the invasive capacity and ligand concentration in control tumors ( Figure 6H). This relationship was slightly lost after anti-Sema4D treatment.

Clinical relevance of Sema4D and SDF1/CXCR4 axis
After demonstrating both in vitro and in vivo the role of Sema4D and SDF1/CXCR4 in tumor malignization of the RIP1Tag2 mouse model, we sought to decipher whether these same mechanisms could be also playing the role in the clinical setting. We found Sema4D expression to be significantly increased in metastatic samples when compared to either primary non-malignant and malignant tumors or normal pancreatic controls ( Figure 7A). Besides, whereas SDF1 expression remained practically unaltered, we found a significant increase in CXCR4 receptor expression between normal and both primary tumor subtypes and metastases ( Figure 7B-C). In fact, there is a gradual increase of CXCR4 that correlates with malignization, thus implying a role for this protein as a tumor progression driver. Furthermore, we evaluated the correlation between Sema4D and CXCR4 expression in nonmalignant (non-metastatic) and malignant (metastatic) primary tumor samples of PanNET patients. Contrary to non-metastatic patients, malignant patients showed higher levels of CXCR4 that showed a correlation with Sema4D ( Figure 7D). We finally validated our results using the human macrophage cell line, THP-1 ( Figure 7E-F). After anti-Sema4D treatment, THP-1 cells demonstrated an increased migratory phenotype and SDF1 release ( Figure 7E-F), without alteration of CXCL12 or CXCR4 expression (Supplementary Figure  14). Overall, the clincal data validates a possible link of SemaD-SDF1/CXCR4 in patient samples of PanNET.

Discussion
Compared to the canonical VEGF, Sema4D is a molecule with quite a different role in angiogenesis since its binding to PlxnB1 can promote different and sometimes opposing cellular responses including vascular guidance (35). Indeed, these differences in vascular targeting potential provide an explanation for the negligible effects in endothelial structures after anti-Sema4D treatment without reduction in MVD and no increased levels of tumor hypoxia. Nevertheless, by PDGF-B reduction anti-Sema4D treatment produced a pericyte structural alteration that functionally modified vessel perfusion and hyperpermeability, thus altering tumor growth.
Moreover, it is widely accepted that a partial inhibition of angiogenesis would not produce an increase of hypoxia within tumours and could not trigger the secondary unwanted proinvasive and malignant effects (27). Contrarily, we have observed that although anti-Sema4D therapy produced a partial effect in vessel functionality, without induction of hypoxia, it still produced the same pro-invasive effect as anti-VEGF therapy in the PanNET model.

Anti-Sema4D promotes tumor invasion via Tumor-associated macrophages
Protumoral roles for Sema4D typically involve tumor cell-derived Sema4D and there is little evidence about the role of stromal Sema4D (36), Interestingly, in RIP1-Tag2 tumors, the main source of Sema4D are macrophages infiltrating the tumor stroma. In recent years, a critical role for tumor microenvironment and particularly TAMs has been demonstrated (37,38). Their contribution to tumor growth and progression has even been reported in the clinical setting, with correlation between a high intratumor TAM content and a poor prognosis (38). In this study we observe an increase in SEMA4D positive macrophages inside tumors and in the invasive front, which goes in agreement with previously published data where Sema4D controls immune cell motility (10)(39). Our knockdown and recombinant Sema4D experiments strongly suggest that the antibody mediates a Sema4Ddependent retrograde signaling engagement in the membrane of macrophages, rather than a function blocking effect. This retrograde signaling has been previously published for this family of transmembrane proteins in different settings (reviewed in 32 and 33). Furthermore, we validated these results utilizing another anti-Sema4D antibody clone 3B4, but not with clone SK-3, which demonstrates these are antibody-specific effects over macrophages.
SDF1/CXCR4 signaling axis has an important role in cancer progression (40). In vitro, we have proven the chemoattractant capacity of SDF1 and its stromal origin, and we have demonstrated that SDF1 release from macrophages is dependent on Sema4D expression and independent of its receptors. In vivo, our results show tumor cells ability to respond to SDF1 stimulus, both in primary tumors and metastases, and the existence of a receptor-ligand positive feedback loop. In fact, the correlation between the invasive capacity of the tumor and SDF1 concentration in control tumors, which is lost after anti-Sema4D treatment, suggests that the SDF1/CXCR4 signaling cascade is already activated regardless of the invasive capacity of the treated tumors.
In the clinical setting, VX15/2503, the humanized anti-Sema4D antibody, showed promising results in the first-in-human phase I clinical trial, with a 45% of patients exhibiting the absence of disease progression for at least 8 weeks (17). Consistently, our own data show anti-Sema4D antibody inhibits tumour growth of PanNETs with a tendency to increase lifespan but also invasion and metastasis. This latter adaptive response to treatment has not been evaluated in patients. Importantly, the combined expression of Sema4D and PlxnB1 is an independent risk factor for disease relapse in colorectal cancer. (41). Other tumors where Sema4D overexpression has been reported as a negative prognostic marker include breast, ovary, soft tissue sarcomas and pancreas (42)(43)(44). Our data demonstrate that both Sema4D and CXCR4 expression increase with tumor progression in PanNETs and also a positive correlation between Sema4D and CXCR4 expression in metastatic PanNET samples. Thus implying that Sema4D and CXCR4 expression are related to the malignization process in patients. Taking into account the inexistence of anti-Sema4D treated PanNET patient samples, these study remarks the role of Sema4D as a potential candidate of tumor malignization in this type of tumors. We have proven, using in vitro, in vivo and in silico approaches, that stromal or immune cells are the primary source of Sema4D, rather than tumor cells.
In conclusion, we describe a hypoxia independent novel mechanism of tumor malignization in the RIP1-Tag2 model, where the signaling engagement of anti-Sema4D antibody binding to Sema4D in macrophages seems to be responsible for the malignant phenotype via SDF1/ CXCR4 signaling axis activation ( Figure 7G). Our study suggests a combinantion of anti-Sema4D therapy and small molecule inhibitors of selected macrophage functions coud be a new therapeutical strategy for PanNET patients. Future studies combining non-traditional anti-angiogenics and novel immunotherapies would undoubtedly shed light into the role of tumor-associated cells, allowing overcoming the undesired resistance.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.   A) Immunohistochemistry staining for Glut1 and pimonidazole (pimo) in untreated (ctrl), anti-Sema4D (α-S4D) and anti-VEGFR2 (α-V2R) treated samples. B) Quantification of the incidence of hypoxic tumors after anti-Sema4D and anti-VEGFR2 1 week treatment compared to controls by staining of pimonidazole adducts (n≥132). C) Quantification by Glut1 staining of the incidence of hypoxic tumors in anti-Sema4D and anti-VEGFR2 2 wks short-term (left) and 4 wks long-term (right) treatments compared to controls (n≥75). B-C. Mann-Whitney test. D) qRT-PCR Taqman analysis of c-met relative to Hprt1 housekeeping gene expression. RNA from RIP1-Tag2 untreated or anti-Sema4D treated mice was analyzed. Mann-Whitney test (n=3). E) Western blot analysis of the active form of c-Met protein (phospho c-met) in control and α-S4D treated RIP1-Tag2 tumors. α-tubulin protein is used as a housekeeping control. Lysate from A549 cells treated with HGF in equal conditions was used as a positive control for c-met phosphorylation. RAW cell line was used as a negative control. F) qRT-PCR Taqman analysis of c-met relative to Hprt1 housekeeping gene expression. RNA from βTC4 cells was analysed and RNA from murine kidney tissue was used as a positive control. G) Western blot analysis of the active form of c-Met protein (phospho c-met) in two independent samples of control, anti-Sema4D (α-S4D) and HGF treated βTC4 cells. α-tubulin protein is used as a housekeeping control. Lysate from A549 cells treated with HGF in equal conditions was used as a positive control for c-met phosphorylation. RAW cell line was used as a negative control.     Results are presented as number of migratory cells per field normalized by the untreated control. Mann-Whitney test (n≥30). F) Quantification of SDF1 protein release by ELISA analysis of conditioned media from control and anti-Sema4D treated THP-1 cells. Results are presented as ng of SDF1 per total protein μg for each condition normalized by the untreated control. Mann-Whitney test (n=3). G) Proposed model for anti-Sema4D derived malignization. Targeting of macrophage derived Sema4D produces macrophage activation and secretion of pro-invasive molecules such as SDF1. Secreted SDF1 is latter bound to its CXCR4 receptor in tumor cells to drive tumor cell invasion. Figure was created using Servier Medical Art according to a Creative Commons Attribution 3.0 Unported License guidelines 3.0 (https://creativecommons.org/licenses/by/3.0/). Simplification and color changes were made to the original cartoons.