Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. DESIGN: We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. RESULTS: PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC. Conclusion PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.

Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown. APPROACH AND RESULTS: We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8+ ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4+ cells. We also found that under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody. CONCLUSIONS: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models.

Anti-angiogenesis for cancer revisited: Is there a role for combinations with immunotherapy?

Angiogenesis is defined as the formation of new blood vessels from preexisting vessels and has been characterized as an essential process for tumor cell proliferation and viability. This has led to the development of pharmacological agents for anti-angiogenesis to disrupt the vascular supply and starve tumor of nutrients and oxygen, primarily through blockade of VEGF/VEGFR signaling. This effort has resulted in 11 anti-VEGF drugs approved for certain advanced cancers, alone or in combination with chemotherapy or other targeted therapies. But this success had only limited impact on overall survival of cancer patients and rarely resulted in durable responses. Given the recent success of immunotherapies, combinations of anti-angiogenics with immune checkpoint blockers have become an attractive strategy. However, implementing such combinations will require a better mechanistic understanding of their interaction. Due to overexpression of pro-angiogenic factors in tumors, their vasculature is often tortuous and disorganized, with excessively branched leaky vessels. This enhances vascular permeability, which in turn is associated with high interstitial fluid pressure, and a reduction in blood perfusion and oxygenation. Judicious dosing of anti-angiogenic treatment can transiently normalize the tumor vasculature by decreasing vascular permeability and improving tumor perfusion and blood flow, and synergize with immunotherapy in this time window. However, anti-angiogenics may also excessively prune tumor vessels in a dose and time-dependent manner, which induces hypoxia and immunosuppression, including increased expression of the immune checkpoint programmed death receptor ligand (PD-L1). This review focuses on revisiting the concept of anti-angiogenesis in combination with immunotherapy as a strategy for cancer treatment.

CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice.

UNLABELLED: Sorafenib, a broad tyrosine kinase inhibitor, is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC) but provides limited survival benefits. Recently, immunotherapy has emerged as a promising treatment strategy, but its role remains unclear in HCCs, which are associated with decreased cytotoxic CD8(+) T-lymphocyte infiltration in both murine and human tumors. Moreover, in mouse models after sorafenib treatment intratumoral hypoxia is increased and may fuel evasive resistance. Using orthotopic HCC models, we now show that increased hypoxia after sorafenib treatment promotes immunosuppression, characterized by increased intratumoral expression of the immune checkpoint inhibitor programmed death ligand-1 and accumulation of T-regulatory cells and M2-type macrophages. We also show that the recruitment of immunosuppressive cells is mediated in part by hypoxia-induced up-regulation of stromal cell-derived 1 alpha. Inhibition of the stromal cell-derived 1 alpha receptor (C-X-C receptor type 4 or CXCR4) using AMD3100 prevented the polarization toward an immunosuppressive microenvironment after sorafenib treatment, inhibited tumor growth, reduced lung metastasis, and improved survival. However, the combination of AMD3100 and sorafenib did not significantly change cytotoxic CD8(+) T-lymphocyte infiltration into HCC tumors and did not modify their activation status. In separate experiments, antibody blockade of the programmed death ligand-1 receptor programmed death receptor-1 (PD-1) showed antitumor effects in treatment-naive tumors in orthotopic (grafted and genetically engineered) models of HCC. However, anti-PD-1 antibody treatment had additional antitumor activity only when combined with sorafenib and AMD3100 and not when combined with sorafenib alone. CONCLUSION: Anti-PD-1 treatment can boost antitumor immune responses in HCC models; when used in combination with sorafenib, anti-PD-1 immunotherapy shows efficacy only with concomitant targeting of the hypoxic and immunosuppressive microenvironment with agents such as CXCR4 inhibitors.

Inhibition of CXCR4 Enhances the Efficacy of Radiotherapy in Metastatic Prostate Cancer Models.

Radiotherapy (RT) is a standard treatment for patients with advanced prostate cancer (PCa). Previous preclinical studies showed that SDF1α/CXCR4 axis could mediate PCa metastasis (most often to the bones) and cancer resistance to RT. We found high levels of expression for both SDF1α and its receptor CXCR4 in primary and metastatic PCa tissue samples. In vitro analyses using PCa cells revealed an important role of CXCR4 in cell invasion but not radiotolerance. Pharmacologic inhibition of CXCR4 using AMD3100 showed no efficacy in orthotopic primary and bone metastatic PCa models. However, when combined with RT, AMD3100 potentiated the effect of local single-dose RT (12 Gy) in both models. Moreover, CXCR4 inhibition also reduced lymph node metastasis from primary PCa. Notably, CXCR4 inhibition promoted the normalization of bone metastatic PCa vasculature and reduced tissue hypoxia. In conclusion, the SDF1α/CXCR4 axis is a potential therapeutic target in metastatic PCa patients treated with RT.

Combining losartan with radiotherapy increases tumor control and inhibits lung metastases from a HER2/neu-positive orthotopic breast cancer model.

BACKGROUND: Patients with metastatic HER2/neu-positive (HER2/neu +) breast cancer (BC) often experience treatment resistance, disease recurrences and metastases. Thus, new approaches for improving the treatment of HER2/neu + BC to prevent metastatic dissemination are urgently needed. Our previous studies have shown that losartan, an angiotensin receptor blocker, increases tumor perfusion and decreases hypoxia in a number of tumor models. Hypoxia reduces the efficacy of radiation and increases metastases. We therefore hypothesized that by modifying tumor stroma and increasing oxygenation, losartan will improve the outcome of radiotherapy and inhibit disease progression in a highly metastatic HER2/neu + murine BC model. METHODS: We established a metastatic HER2/neu + murine BC line (MCa-M3C) and used it to generate mammary fat pad isografts in syngeneic female FVB/N mice. Starting on day 3 after orthotopic tumor implantation, we administered a 7-day losartan treatment (40 mg/kg BW, gavage daily); or a 7-day losartan treatment followed by 20 Gy single dose local irradiation (S-IR) on day 10 (tumor size ~ 100 mm3), or 20 Gy local fractionated (5 × 4 Gy daily) irradiation (F-IR) on days 10-14. We analyzed tumor-growth delay (TGD), development of spontaneous lung metastases, animal survival, tumor vascular density, and tumor hypoxia. RESULTS: Treatments with S-IR, F-IR, Losartan + S-IR, or Losartan + F-IR resulted in a significantly increased TGD (8-16 days) in MCa-M3C tumors versus controls. However, the combination of Losartan + S-IR and Losartan + F-IR further enhanced tumor response to radiation alone by increasing TGD an additional 5 to 8 days for both single and fractionated dose irradiation (P < 0.01), decreasing lung metastasis (Losartan + IR vs. Control, P < 0.025), and increasing animal survival (Losartan + IR vs. Control, P = 0.0303). In addition, losartan treatment significantly increased tumor vascularity (P = 0.0314) and decreased pimonidazole positive (hypoxic) area (P = 0.0002). CONCLUSIONS: Combining losartan with local irradiation significantly enhanced tumor response, at least in part via reduced tumor hypoxia presumably due to increased tumor perfusion. Our findings suggest that combining losartan with radiotherapy is a potential new treatment strategy for local control and inhibiting metastasis in HER2 + BC.

Regorafenib combined with PD1 blockade increases CD8 T-cell infiltration by inducing CXCL10 expression in hepatocellular carcinoma.

BACKGROUND AND PURPOSE: Combining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms of benefit remain unclear. We examined the efficacy and defined the mechanisms of benefit when combining regorafenib (a multikinase antivascular endothelial growth factor receptor inhibitor) with PD1 blockade in murine hepatocellular carcinoma (HCC) models. BASIC PROCEDURES: We used orthotopic models of HCC in mice with liver damage to test the effects of regorafenib-dosed orally at 5, 10 or 20 mg/kg daily-combined with anti-PD1 antibodies (10 mg/kg intraperitoneally thrice weekly). We evaluated the effects of therapy on tumor vasculature and immune microenvironment using immunofluorescence, flow cytometry, RNA-sequencing, ELISA and pharmacokinetic/pharmacodynamic studies in mice and in tissue and blood samples from patients with cancer. MAIN FINDINGS: Regorafenib/anti-PD1 combination therapy increased survival compared with regofarenib or anti-PD1 alone in a regorafenib dose-dependent manner. Combination therapy increased regorafenib uptake into the tumor tissues by normalizing the HCC vasculature and increasing CD8 T-cell infiltration and activation at an intermediate regorafenib dose. The efficacy of regorafenib/anti-PD1 therapy was compromised in mice lacking functional T cells (Rag1-deficient mice). Regorafenib treatment increased the transcription and protein expression of CXCL10-a ligand for CXCR3 expressed on tumor-infiltrating lymphocytes-in murine HCC and in blood of patients with HCC. Using Cxcr3-deficient mice, we demonstrate that CXCR3 mediated the increased intratumoral CD8 T-cell infiltration and the added survival benefit when regorafenib was combined with anti-PD1 therapy. PRINCIPAL CONCLUSIONS: Judicious regorafenib/anti-PD1 combination therapy can inhibit tumor growth and increase survival by normalizing tumor vasculature and increasing intratumoral CXCR3+CD8 T-cell infiltration through elevated CXCL10 expression in HCC cells.

Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy.

Several cancer immunotherapy approaches, such as immune checkpoint blockade and adoptive T-cell therapy, boost T-cell activity against the tumor, but these strategies are not effective in the absence of T cells specific for displayed tumor antigens. Here we outline an immunotherapy in which endogenous T cells specific for a noncancer antigen are retargeted to attack tumors. The approach relies on the use of antibody-peptide epitope conjugates (APECs) to deliver suitable antigens to the tumor surface for presention by HLA-I. To retarget cytomegalovirus (CMV)-specific CD8+ T cells against tumors, we used APECs containing CMV-derived epitopes conjugated to tumor-targeting antibodies via metalloprotease-sensitive linkers. These APECs redirect pre-existing CMV immunity against tumor cells in vitro and in mouse cancer models. In vitro, APECs activated specifically CMV-reactive effector T cells whereas a bispecific T-cell engager activated both effector and regulatory T cells. Our approach may provide an effective alternative in cancers that are not amenable to checkpoint inhibitors or other immunotherapies.

Overcoming sorafenib evasion in hepatocellular carcinoma using CXCR4-targeted nanoparticles to co-deliver MEK-inhibitors.

Sorafenib is a RAF inhibitor approved for several cancers, including hepatocellular carcinoma (HCC). Inhibition of RAF kinases can induce a dose-dependent "paradoxical" upregulation of the downstream mitogen-activated protein kinase (MAPK) pathway in cancer cells. It is unknown whether "paradoxical" ERK activation occurs after sorafenib therapy in HCC, and if so, if it impacts the therapeutic efficacy. Here, we demonstrate that RAF inhibition by sorafenib rapidly leads to RAF dimerization and ERK activation in HCCs, which contributes to treatment evasion. The transactivation of RAF dimers and ERK signaling promotes HCC cell survival, prevents apoptosis via downregulation of BIM and achieves immunosuppression by MAPK/NF-kB-dependent activation of PD-L1 gene expression. To overcome treatment evasion and reduce systemic effects, we developed CXCR4-targeted nanoparticles to co-deliver sorafenib with the MEK inhibitor AZD6244 in HCC. Using this approach, we preferentially and efficiently inactivated RAF/ERK, upregulated BIM and down-regulated PD-L1 expression in HCC, and facilitated intra-tumoral infiltration of cytotoxic CD8+ T cells. These effects resulted in a profound delay in tumor growth. Thus, this nano-delivery strategy to selectively target tumors and prevent the paradoxical ERK activation could increase the feasibility of dual RAF/MEK inhibition to overcome sorafenib treatment escape in HCC.

The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

An orthotopic mouse model of hepatocellular carcinoma with underlying liver cirrhosis.

Subcutaneous xenografts have been used for decades to study hepatocellular carcinoma (HCC). These models do not reproduce the specific pathophysiological features of HCCs, which occur in cirrhotic livers that show pronounced necroinflammation, abnormal angiogenesis and extensive fibrosis. As these features are crucial for studying the role of the pathologic host microenvironment in tumor initiation, progression and treatment response, alternative HCC models are desirable. Here we describe a syngeneic orthotopic HCC model in immunocompetent mice with liver cirrhosis induced by carbon tetrachloride (CCl4) that recapitulates key features of human HCC. Induction of substantial hepatic fibrosis requires 12 weeks of CCl4 administration. Intrahepatic implantation of mouse HCC cell lines requires 30 min per mouse. Tumor growth varies by tumor cell line and mouse strain used. Alternatively, tumors can be induced in a genetically engineered mouse model. In this setting, CCl4 is administered for 12 weeks after tail-vein injection of Cre-expressing adenovirus (adeno-Cre) in Stk4(-/-)Stk3(F/-) (also known as Mst1(-/-)Mst2(F/-); F indicates a floxed allele) mice, and it results in the development of HCC tumors (hepatocarcinogenesis) concomitantly with liver cirrhosis.