PI3Kγ is a molecular switch that controls immune suppression.

Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPß activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPß activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.

Injection of SVF combined with HBO2 improves viability of unfavorably designed flaps.

PURPOSE: Soft-tissue reconstruction is complicated by ischemia and reperfusion injury. Animal trials have documented the independent healing benefits of hyperbaric oxygen preconditioning (HBOP) and stem cell delivery in cutaneous flaps. We explored the role of HBOP and stem cell delivery in flap preconditioning and survival. METHODS: We designed a randomized controlled trial to assess the effects of hyperbaric oxygen preconditioning and stromal vascular fraction (SVF) delivery on flap survival. Of the first 24 guinea pigs, six received neither HBOP nor injections, and six underwent HBOP without injections. Of the remaining 12 animals, six received SVF or saline injections in the absence of HBOP. The final six animals received autologous SVF injections or saline injections followed by four HBOP treatments. To enhance clinical relevance, a group of 6 animals underwent HBOP prior to SVF or saline injections. Thereafter, an unfavorably designed cutaneous flap was elevated and assessed via study-blinded observer, as well as by quantification of TUNEL-positive cells. RESULTS: Distal necrosis of the tissue flap was more extensive in the no-intervention group (45% of flap). Flaps treated with HBOP only and those treated with SVF injections demonstrated only 38.2% and 27.1% distal necrosis. The most significant difference occurred in the combination HBOP and SVF group, where distal necrosis was only 21.1% of the flap (p ≤ 0.05). SVF delivery immediately prior to flap elevation further minimized distal necrosis of the flap to 15.6%. These findings were mirrored by the TUNEL assay. CONCLUSIONS: Combining HBOP and SVF improves flap viability.

PI3Kα activates integrin α4ß1 to establish a metastatic niche in lymph nodes.

Lymph nodes are initial sites of tumor metastasis, yet whether the lymph node microenvironment actively promotes tumor metastasis remains unknown. We show here that VEGF-C/PI3Kα-driven remodeling of lymph nodes promotes tumor metastasis by activating integrin α4ß1 on lymph node lymphatic endothelium. Activated integrin α4ß1 promotes expansion of the lymphatic endothelium in lymph nodes and serves as an adhesive ligand that captures vascular cell adhesion molecule 1 (VCAM-1)(+) metastatic tumor cells, thereby promoting lymph node metastasis. Experimental induction of α4ß1 expression in lymph nodes is sufficient to promote tumor cell adhesion to lymphatic endothelium and lymph node metastasis in vivo, whereas genetic or pharmacological blockade of integrin α4ß1 or VCAM-1 inhibits it. As lymph node metastases accurately predict poor disease outcome, and integrin α4ß1 is a biomarker of lymphatic endothelium in tumor-draining lymph nodes from animals and patients, these results indicate that targeting integrin α4ß1 or VCAM to inhibit the interactions of tumor cells with the lymph node microenvironment may be an effective strategy to suppress tumor metastasis.

Correlations between macrophage/microglial activation marker sTREM-2 and measures of T-cell activation, neuroaxonal damage and disease severity in multiple sclerosis.

BACKGROUND: Soluble triggering receptor expressed on myeloid cells-2 (sTREM-2) is a marker of macrophage and microglial activation and is increased in the cerebrospinal fluid (CSF) in multiple sclerosis (MS). OBJECTIVE: To determine the relationships among sTREM-2, T cell activation, neuroaxonal damage and clinical features of MS. METHODS: Enzyme-linked immunosorbent assays were used to measure the levels of sTREM-2, soluble CD27 (sCD27, a marker of T cell activation), neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) in the CSF of 42 patients with MS (including nine with clinically isolated syndrome) and 15 patients with other neurological diseases (OND) and in the serum of 164 patients with MS, 87 patients with OND and 62 healthy controls. RESULTS: sTREM-2 was significantly elevated in the CSF (p = 0.012), but not in the serum, in MS compared to OND. In MS, CSF sTREM-2 correlated positively with CSF sCD27 (p = 0.005), CSF NfL (p = 0.0001), CSF pNfH (p = 0.0006), Expanded Disability Status Scale (EDSS) score (p = 0.0079) and MS Severity Score (MSSS) (p = 0.0006). CONCLUSION: In MS the level of sTREM-2 in the CSF is related to measures of T cell activation (sCD27), neuroaxonal damage (NfL and pNfH), disability (EDSS) and disease severity (MSSS).

PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Coadministration of endothelial and smooth muscle progenitor cells enhances the efficiency of proangiogenic cell-based therapy.

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 10(6)). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.

Preclinical assessment of safety and efficacy of intravenous delivery of autologous adipose-derived regenerative cells (ADRCs) in the treatment of severe thermal burns using a porcine model.

OBJECTIVE: A number of studies have reported that application of autologous adipose-derived cell populations leads to improved outcome in different preclinical models of thermal burn injury. However, these studies were limited to assessment of relatively small injuries amounting to only ∼2% of total body surface area (TBSA) in which the complications associated with large burns (e.g.: systemic inflammation and the need for fluid resuscitation) are absent. In anticipation of translating this approach to a clinical trial in which these complications would be present we applied a preclinical model that more closely resembles a patient with large thermal burn injury requiring skin grafting. Thus, the present study used a porcine model to investigate safety and efficacy of intravenous delivery of ADRCs in the treatment of a complex burn injury comprising ∼20% TBSA and including both moderately deep (44%) partial and full thickness burns, and the injury associated with skin graft harvest. METHODS: Two pairs of full thickness and partial thickness burns involving in total ∼20% TBSA were created on the back of Yorkshire pigs (n=15). Three days post-burn, full thickness wounds were excised and grafted with a 3:1 meshed autologous split thickness skin graft (STSG). Partial thickness wounds were not treated other than with dressings. Animals were then randomized to receive intravenous delivery of ADRCs (n=8) or vehicle control (n=7). Safety was assessed by monitoring systemic parameters (blood gases, hematology, and clinical chemistry) throughout the course of the study. Wound healing for both types of burn wound and for the skin graft donor sites was followed for 18days using wound imaging, histology, and trans-epidermal water loss (TEWL; skin barrier function assessment). RESULTS: No serious adverse events related to ADRC infusion were noted in any of the animals. Delivery of ADRCs appeared to be safe with none of the systemic safety parameters worsened compared to the control group. TEWL and histological analyses revealed that ADRC treatment was associated with significantly accelerated healing of skin graft (27.1% vs. 1.1% on Day 5 post-grafting), donor site (52.8% vs. 33.1% on Day 5 post-excision) and partial thickness burn (81.8% vs. 59.8% on Day 18 post-treatment). Data also suggested that ADRC treatment improved parameters associated with skin graft elasticity. CONCLUSIONS: This study demonstrated that intravenous delivery of autologous ADRCs appears to be a safe and feasible approach to the treatment of large burns and supports the use of ADRCs as an adjunct therapy to skin grafting in patients with severe burns.

Increase in vascular permeability and vasodilation are critical for proangiogenic effects of stem cell therapy.

BACKGROUND: Proangiogenic cell therapy based on administration of bone marrow-derived mononuclear cells (BMCs) or endothelial progenitor cells (EPCs) is now under investigation in humans for the treatment of ischemic diseases. However, mechanisms leading to the beneficial effects of BMCs and EPCs remain unclear. METHODS AND RESULTS: BMC- and CD34+-derived progenitor cells interacted with ischemic femoral arteries through SDF-1 and CXCR4 signaling and released nitric oxide (NO) via an endothelial nitric oxide synthase (eNOS)-dependent pathway. BMC-induced NO production promoted a marked vasodilation and disrupted vascular endothelial-cadherin/beta-catenin complexes, leading to increased vascular permeability. NO-dependent vasodilation and hyperpermeability were critical for BMC infiltration in ischemic tissues and their proangiogenic potential in a model of hindlimb ischemia in mice. CONCLUSIONS: Our results propose a new concept that proangiogenic progenitor cell activity does not rely only on their ability to differentiate into endothelial cells but rather on their capacity to modulate the function of preexisting vessels.

PI3Kγ Activates Integrin α4 and Promotes Immune Suppressive Myeloid Cell Polarization during Tumor Progression.

Immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) accumulate in tumors where they inhibit T cell-mediated antitumor immune responses and promote tumor progression. Myeloid cell PI3Kγ plays a role in regulating tumor immune suppression by promoting integrin α4-dependent MDSC recruitment to tumors and by stimulating the immunosuppressive polarization of MDSCs and TAMs. Here, we show that integrin α4 promotes immunosuppressive polarization of MDSCs and TAMs downstream of PI3Kγ, thereby inhibiting antitumor immunity. Genetic or pharmacological suppression of either PI3Kγ or integrin α4 blocked MDSC recruitment to tumors and also inhibited immune suppressive myeloid cell polarization, thereby reducing expression of IL10 and increasing expression of IL12 and IFNγ within tumors. Inhibition of PI3Kγ or integrin α4 within tumors stimulated dendritic cell and CD8+ T-cell recruitment and maturation, as well as tumor cell cytotoxicity in vivo, thereby inhibiting tumor growth. As blockade of PI3Kγ or integrin α4 prevents accumulation of MDSC and reduces myeloid cell expression of immunosuppressive factors that stimulate tumor immune escape, these results highlight PI3Kγ and integrin α4 as targets for the design of cancer therapeutics. Cancer Immunol Res; 5(11); 957-68. ©2017 AACR.

Autologous adipose-derived regenerative cell therapy modulates development of hypertrophic scarring in a red Duroc porcine model.

BACKGROUND: Effective prevention and treatment of hypertrophic scars (HTSs), a common consequence of deep-partial thickness injury, remain a significant clinical challenge. Previous studies from our group have shown that autologous adipose-derived regenerative cells (ADRCs) represent a promising approach to improve wound healing and, thereby, impact HTS development. The purpose of this study was to assess the influence of local delivery of ADRCs immediately following deep-partial thickness cutaneous injury on HTS development in the red Duroc (RD) porcine model. METHODS: Bilateral pairs of deep-partial thickness excisional wounds (2 mm depth; 58 cm2 area) were created using an electric dermatome on RD pigs (n = 12). Autologous ADRCs were isolated from the inguinal fat pad and then sprayed directly onto the wound at a dose of 0.25 × 106 viable cells/cm2. The paired contralateral wound received vehicle control. Wound healing and development of HTS were assessed over 6 months using digital imaging, quantitative measurement of skin hardness and pigmentation, and histology. RESULTS: Data showed that ADRC treatment led to reduced scar hyperpigmentation compared to control (p < 0.05). Using the Durometer, at 2 and 6 months post-injury, skin hardness was 10-20% lower in ADRCs-treated wounds compared to control vehicle (p < 0.05). A similar trend was observed with the skin fibrometer. ADRC treatment promoted more normal collagen organization, improvement in the number of rete ridges (p < 0.01), longer elastic fiber length (p < 0.01), and reduced hypervascularity (blood vessel density; p < 0.05). ADRC treatment was associated with modulation of IL-6 expression within the wound/scar with upregulation 2 weeks after injury (wound healing phase) and downregulation at 2 months (early scarring phase) post-treatment compared to control CONCLUSIONS: These findings support the potential therapeutic value of autologous ADRC administration for reduction of HTS development following deep-partial cutaneous injury.

Development of a combined radiation and full thickness burn injury minipig model to study the effects of uncultured adipose-derived regenerative cell therapy in wound healing.

PURPOSE: To develop an approach that models the cutaneous healing that occurs in a patient with full thickness thermal burn injury complicated by total body radiation exposure sufficient to induce sub-lethal prodromal symptoms. An assessment of the effects of an autologous cell therapy on wound healing on thermal burn injury with concomitant radiation exposure was used to validate the utility of the model. METHODS: Göttingen minipigs were subjected to a 1.2 Gy total body irradiation by exposure to a 6 MV X-ray linear accelerator followed by ∼10 cm2 full thickness burns (pre-heated brass block with calibrated spring). Three days after injury, wounds were excised to the underlying fascia and each animal was randomized to receive treatment with autologous adipose-derived regenerative cells (ADRC) delivered by local or intravenous injection, or vehicle control. Blood counts were used to assess radiation-induced marrow suppression. All animals were followed using digital imaging to assess wound healing. Full-thickness biopsies were obtained at 7, 14, 21 and 30 days' post-treatment. RESULTS: Compared to animals receiving burn injury alone, significant transient neutropenia and thrombocytopenia were observed in irradiated subjects with average neutrophil nadir of 0.79 × 103/µl (day 15) and platelet nadir of 60 × 103/µl (day 12). Wound closure through a combination of contraction and epithelialization from the wound edges occurred over a period of approximately 28 days' post excision and treatment. Re-epithelialization was accelerated in wounds treated with ADRC (mean 3.5-fold increase at 2 weeks post-treatment relative to control). This acceleration was accompanied by an average 67% increase in blood vessel density and 30% increase in matrix (collagen) deposition. Similar results were observed when ADRC were injected either directly into the wound or by intravenous administration. CONCLUSIONS: Although preliminary, this study provides a reproducible minipig model of thermal burn injury complicated by myelosuppressive total body irradiation that utilizes standardized procedures to evaluate novel countermeasures for potential use following attack by an improvised nuclear device.

Adipose-Derived Regenerative Cell Therapy for Burn Wound Healing: A Comparison of Two Delivery Methods.

Objective: The use of noncultured autologous stromal vascular fraction or clinical grade adipose-derived regenerative cells (ADRCs) is a promising strategy to promote wound healing and tissue repair. Nevertheless, issues regarding the optimal mode of administration remain unclear. The purpose of this study was to compare the effects of local injection and topical spray delivery of ADRCs in a porcine model of thermal burns. Approach: Full-thickness thermal burns were created on the dorsum of 10 Gottingen minipigs. Two days following injury, wounds underwent fascial excision and were randomized to receive control vehicle or freshly isolated autologous ADRCs delivered by either multiple injections into or surrounding the wound bed, or by spray onto the wound surface (0.25 × 106 viable cells/cm2). Healing was evaluated by planimetry, histopathology, and immunohistochemistry at day 7, 12, 16, 21, and 28 posttreatment. Results:In vitro analysis demonstrated that there was no substantial loss of cell number or viability attributable to the spray procedure. Planimetric assessment revealed that delivery of ADRCs by either local injection or topical spray increased wound reepithelialization relative to control at day 14. No significant difference in wound reepithelialization was observed between both delivery approaches. In addition, on day 7 posttreatment, blood vessel density was greater in wounds receiving local or topical spray ADRCs than in the wounds treated with vehicle control. Histopathologic analysis suggests that ADRC treatment may modulate the inflammatory response by reducing neutrophil infiltration at day 7 and 12 posttreatment, irrespective of the route of administration. Conclusions: These data demonstrate that local injection and spray delivery of ADRCs modulate inflammation and improve wound angiogenesis and epithelialization. Importantly, both delivery routes exhibited similar effects on wound healing. Given the greater ease-of-use associated with topical spray delivery, these data support the use of a spray system for autologous ADRC delivery.

Macrophage PI3Kγ Drives Pancreatic Ductal Adenocarcinoma Progression.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a low 5-year survival rate, yet new immunotherapeutic modalities may offer hope for this and other intractable cancers. Here, we report that inhibitory targeting of PI3Kγ, a key macrophage lipid kinase, stimulates antitumor immune responses, leading to improved survival and responsiveness to standard-of-care chemotherapy in animal models of PDAC. PI3Kγ selectively drives immunosuppressive transcriptional programming in macrophages that inhibits adaptive immune responses and promotes tumor cell invasion and desmoplasia in PDAC. Blockade of PI3Kγ in PDAC-bearing mice reprograms tumor-associated macrophages to stimulate CD8(+) T-cell-mediated tumor suppression and to inhibit tumor cell invasion, metastasis, and desmoplasia. These data indicate the central role that macrophage PI3Kγ plays in PDAC progression and demonstrate that pharmacologic inhibition of PI3Kγ represents a new therapeutic modality for this devastating tumor type. SIGNIFICANCE: We report here that PI3Kγ regulates macrophage transcriptional programming, leading to T-cell suppression, desmoplasia, and metastasis in pancreas adenocarcinoma. Genetic or pharmacologic inhibition of PI3Kγ restores antitumor immune responses and improves responsiveness to standard-of-care chemotherapy. PI3Kγ represents a new therapeutic immune target for pancreas cancer. Cancer Discov; 6(8); 870-85. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Uncultured adipose-derived regenerative cells (ADRCs) seeded in collagen scaffold improves dermal regeneration, enhancing early vascularization and structural organization following thermal burns.

OBJECTIVE: Advances in tissue engineering have yielded a range of both natural and synthetic skin substitutes for burn wound healing application. Long-term viability of tissue-engineered skin substitutes requires the formation and maturation of neo-vessels to optimize survival and biointegration after implantation. A number of studies have demonstrated the capacity of Adipose Derived Regenerative Cells (ADRCs) to promote angiogenesis and modulate inflammation. On this basis, it was hypothesized that adding ADRCs to a collagen-based matrix (CBM) (i.e. Integra) would enhance formation and maturation of well-organized wound tissue in the setting of acute thermal burns. The purpose of this study was to evaluate whether seeding uncultured ADRCs onto CBM would improve matrix properties and enhance healing of the grafted wound. METHODS: Full thickness thermal burns were created on the backs of 8 Gottingen mini-swine. Two days post-injury wounds underwent fascial excision and animals were randomized to receive either Integra seeded with either uncultured ADRCs or control vehicle. Wound healing assessment was performed by digital wound imaging, histopathological and immunohistochemical analyses. RESULTS: In vitro analysis demonstrated that freshly isolated ADRCs adhered and propagated on the CBM. Histological scoring revealed accelerated maturation of wound bed tissue in wounds receiving ADRCs-loaded CBM compared to vehicle-loaded CBM. This was associated with a significant increase in depth of the wound bed tissue and collagen deposition (p<0.05). Blood vessel density in the wound bed was 50% to 69.6% greater in wounds receiving ADRCs-loaded CBM compared to vehicle-loaded CBM (p=0.05) at day 14 and 21. In addition, ADRCs delivered with CBM showed increased blood vessel lumen area and blood vessel maturation at day 21(p=0.05). Interestingly, vascularity and overall cellularity within the CBM were 50% and 45% greater in animals receiving ADRC loaded scaffolds compared to CBM alone (p<0.05). CONCLUSIONS: These data demonstrate that seeding uncultured ADRCs onto CBM dermal substitute enhances wound angiogenesis, blood vessel maturation and matrix remodeling.

Strategies to Enhance the Efficiency of Endothelial Progenitor Cell Therapy by Ephrin B2 Pretreatment and Coadministration with Smooth Muscle Progenitor Cells on Vascular Function During the Wound-Healing Process in Irradiated or Nonirradiated Condition.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization. We therefore assessed the effect of a therapeutic strategy based on EPC administration in the healing of radiation-induced damage. To improve cell therapy for clinical use, we used pretreatment with ephrin B2-Fc (Eph-B2-Fc) and/or coadministration with smooth muscle progenitor cells. At day 3, EPCs promoted dermal wound healing in both nonirradiated and irradiated mice by 1.2- and 1.15-fold, respectively, compared with animals injected with phosphate-buffered saline. In addition, EPCs also improved skin-blood perfusion and capillary density in both irradiated and nonirradiated mice compared with PBS-injected animals. We also demonstrated that activation with Eph-B2-Fc increased wound closure by 1.6-fold compared with unstimulated EPCs in nonirradiated mice. Interestingly, the beneficial effect of Eph-B2-Fc was abolished in irradiated animals. In addition, we found that Eph-B2-Fc stimulation did not improve EPC-induced vascular permeability or adhesiveness compared to unstimulated EPCs. We hypothesized that this effect was due to high oxidative stress during irradiation, leading to inhibition of EPCs' beneficial effect on vascular function. In this line, we demonstrated that, in irradiated conditions, N-acetyl-l-cysteine treatment restored the beneficial effect of EPC stimulation with Eph-B2-Fc in the wound healing process. In conclusion, stimulation by Eph-B2-Fc improved the beneficial effect of EPCs in physiological conditions and irradiated conditions only in association with antioxidant treatment. Additionally, cotherapy was beneficial in pathological conditions.

Integrins in tumor angiogenesis and lymphangiogenesis.

Angiogenesis, the formation of new blood vessel, plays an important role for the growth and metastasis of malignant tumors. The recent identification of specific growth factors for lymphatic vessels and of new lymphatic-specific markers provided evidence for an active role of the lymphatic system during the tumor growth and metastasis processes. Tumor lymphangiogenesis has been shown to play a role in promoting tumor growth and metastasis of tumor cells to distant sites. Integrins play keys roles in the regulation of angiogenesis and lymphangiogenesis during normal development and several diseases. Indeed, integrins control vascular and lymphatic endothelial cell adhesion, migration, and survival. Importantly, integrin inhibitors can block angiogenesis and lymphangiogenesis. In this chapter, we will highlight the role of integrins during angiogenesis and lymphangiogenesis as well as the function of individual integrins during vascular development, postnatal angiogenesis, and lymphangiogenesis. We discuss the role of integrins as potential therapeutic targets for the control of tumor angiogenesis, lymphangiogenesis, and metastatic spread in the treatment of cancer. We also describe methods to analyze expression and function of integrins during angiogenesis and lymphangiogenesis.

Combined blockade of integrin-α4ß1 plus cytokines SDF-1α or IL-1ß potently inhibits tumor inflammation and growth.

Tumor-associated macrophages promote tumor growth by stimulating angiogenesis and suppressing antitumor immunity. Thus, therapeutics that inhibit macrophage recruitment to tumors may provide new avenues for cancer therapy. In this study, we showed how chemoattractants stromal cell-derived growth factor 1 alpha (SDF-1α) and interleukin 1 beta (IL-1ß) collaborate with myeloid cell integrin-α4ß1 to promote tumor inflammation and growth. We found that SDF-1α and IL-1ß are highly expressed in the microenvironments of murine lung, pancreatic, and breast tumors; surprisingly, SDF-1α was expressed only by tumor cells, whereas IL-1ß was produced only by tumor-derived granulocytes and macrophages. In vivo, both factors directly recruited proangiogenic macrophages to tissues, whereas antagonists of both factors suppressed tumor inflammation, angiogenesis, and growth. Signals induced by IL-1ß and SDF-1α promoted the interaction of talin and paxillin with the cytoplasmic tails of integrin-α4ß1, thereby stimulating myeloid cell adhesion to endothelium in vitro and in vivo. Inhibition of integrin-α4ß1, SDF-1α, or IL-1ß was sufficient to block tumor inflammation and growth, and the combined blockade of these molecules greatly accentuated these effects. Furthermore, antagonists of integrin-α4ß1 inhibited chemotherapy-induced tumor inflammation and acted synergistically with chemotherapeutic agents to suppress tumor inflammation and growth. These results show that targeting myeloid cell recruitment mechanisms can be an effective approach to suppress tumor progression.

Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression.

Tumor inflammation promotes angiogenesis, immunosuppression, and tumor growth, but the mechanisms controlling inflammatory cell recruitment to tumors are not well understood. We found that a range of chemoattractants activating G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and Toll-like/IL-1 receptors (TLR/IL1Rs) unexpectedly initiate tumor inflammation by activating the PI3-kinase isoform p110γ in Gr1+CD11b+ myeloid cells. Whereas GPCRs activate p110γ in a Ras/p101-dependent manner, RTKs and TLR/IL1Rs directly activate p110γ in a Ras/p87-dependent manner. Once activated, p110γ promotes inside-out activation of a single integrin, α4ß1, causing myeloid cell invasion into tumors. Pharmacological or genetic blockade of p110γ suppressed inflammation, growth, and metastasis of implanted and spontaneous tumors, revealing an important therapeutic target in oncology.

Integrin alpha4beta1 signaling is required for lymphangiogenesis and tumor metastasis.

Recent studies have shown that lymphangiogenesis or the growth of lymphatic vessels at the periphery of tumors promotes tumor metastasis to lymph nodes. We show here that the fibronectin-binding integrin alpha4beta1 and its ligand fibronectin are novel functional markers of proliferative lymphatic endothelium. Tumors and lymphangiogenic growth factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGF-A, induce lymphatic vessel expression of integrin alpha4beta1. Integrin alpha4beta1 then promotes growth factor and tumor-induced lymphangiogenesis, as genetic loss of integrin alpha4beta1 expression in Tie2Cre+ alpha4(loxp/loxp) mice or genetic loss of alpha4 signaling in alpha4Y991A knock-in mice blocks growth factor and tumor-induced lymphangiogenesis, as well as tumor metastasis to lymph nodes. In addition, antagonists of integrin alpha4beta1 suppress lymphangiogenesis and tumor metastasis. Our studies show that integrin alpha4beta1 and the signals it transduces regulate the adhesion, migration, invasion, and survival of proliferating lymphatic endothelial cells. As suppression of alpha4beta1 expression, signal transduction, or function in tumor lymphatic endothelium not only inhibits tumor lymphangiogenesis but also prevents metastatic disease, these results show that integrin alpha4beta1-mediated tumor lymphangiogenesis promotes metastasis and is a useful target for the suppression of metastatic disease.