Intrinsic and evasive antiangiogenic drug (AAD) resistance is frequently developed in cancer patients, and molecular mechanisms underlying AAD resistance remain largely unknown. Here we describe AAD-triggered, lipid-dependent metabolic reprogramming as an alternative mechanism of AAD resistance. Unexpectedly, tumor angiogenesis in adipose and non-adipose environments is equally sensitive to AAD treatment. AAD-treated tumors in adipose environment show accelerated growth rates in the presence of a minimal number of microvessels. Mechanistically, AAD-induced tumor hypoxia initiates the fatty acid oxidation metabolic reprogramming and increases uptake of free fatty acid (FFA) that stimulates cancer cell proliferation. Inhibition of carnitine palmitoyl transferase 1A (CPT1) significantly compromises the FFA-induced cell proliferation. Genetic and pharmacological loss of CPT1 function sensitizes AAD therapeutic efficacy and enhances its anti-tumor effects. Together, we propose an effective cancer therapy concept by combining drugs that target angiogenesis and lipid metabolism.

In this work, we uncover an alternative mechanism of AAD resistance by lipid metabolism reprogramming. AAD-triggered oxygen and nutrient depletion through suppression of angiogenesis switches the glucose-dependent metabolism to lipid-dependent metabolism, the latter metabolic pathway warrants tumor growth in the presence of a minimal number of microvessels. Our findings propose a concept of an effective therapy by combining AAD and drugs targeting the lipid metabolic pathways.

Unlike most healthy cells, cancer cells exhibit distinctive features of uncontrolled cell proliferation (). To cope with the unlimited growth, expansion, and dissemination, cancer cells must efficiently produce energy, even in poorly oxygenated and nutrient-scarce microenvironments (). Cancer cells show exacerbated glucose uptake and glycolysis-dependent metabolism (i.e., the Warburg effect;). In addition, malignant cells also rely on glutamine consumption to obtain carbon, amino-nitrogen for producing nucleotides, amino acids, and lipid biosynthesis. Recent studies show that highly proliferative cancer cells have lipogenic activity by uptake of exogenous lipids and activating endogenous lipid biosynthesis (). Utilization of exogenous free fatty acids (FFAs) for energy production through the fatty acid oxidation (FAO) metabolic pathway is prominent in non-glycolytic cancers such as prostate cancer and B cell lymphoma (). Several lipogenic enzymes, including acetyl coenzyme A carboxylase and fatty acid synthase (FASN), are often increased in invasive tumors and their expression levels correlate with poor prognosis (). The FAO-limiting enzyme, CPT1 (A and C types), is often overexpressed in many human tumors (). It is known that adipose tissue and FFA significantly contribute to cancer cell survival, proliferation, and migration (). Previously published work also showed that anti-VEGF treatment and tissue hypoxia increase lipid transport and storage through an HIF-1α-dependent mechanism in cancer cells ().

Tumor tissues often experience hypoxia owing to accelerated growth rates of malignant cells, accumulation of metabolic products, disorganization of tumor blood vessels, and high interstitial fluid pressures (). In response to AAD treatment, tumor vascular density often decreases to an extremely low level, creating an elevated hypoxic environment (). It is known that tumor hypoxia can exacerbate expression levels of growth factors and cytokines, which circumvent the drug targets and create possible resistance (). Hypoxia may also change the composition of various cell types within the tumor microenvironment (TME), leading to alteration of cancer invasiveness and drug responses ().

Drugs targeting tumor blood vessels are commonly used in cancer patients and they generally produce limited therapeutic benefits for survival improvement (). One of the main hitches of low therapeutic efficacy is that cancer patients often develop resistance in response to antiangiogenic drug (AAD) treatment (). Patients with cancers grown in organs adjacent to adipose tissues, including breast cancer, prostate cancer, pancreatic cancer, and hepatocellular carcinoma (HCC), show particularly low benefits from antiangiogenic therapy. These cancers located adjacent to adipose tissues often show intrinsic or acquired resistance to antiangiogenic therapy. For example, most patients with pancreatic ductal adenocarcinoma (PDAC) show intrinsic resistance and colorectal cancer (CRC) patients exhibit evasive resistance to bevacizumab (). A puzzling observation in the field of antiangiogenic cancer therapy has been the inconsistency of drug effects in preclinical animal models and in cancer patients (). While most AADs produce overwhelming anti-tumor effects in mouse models, the same drug often lacks anti-cancer effect in human patients. Among numerous possible reasons, the location of tumor implantation in animal models is often different from clinical situations. For example, subcutaneous implantation is a common location for studying animal tumors for the sake of convenience in monitoring tumor growth. However, human tumors rarely originate from a subcutaneous location.

In addition to genetic approaches, we took a pharmacological approach to inhibit CPT1 function using etomoxir as an inhibitor (). Again, treatment of steatotic liver CRCs with a combination of etomoxir and AAD markedly increased anti-cancer effects ( Figures 7 A and 7D ). Luminescent imaging analysis of tumor-bearing livers further validated the enhanced anti-tumor effects of combination therapy ( Figure 7 A). In fact, the combination therapy produced the remarkable effects of suppressing tumor growth ( Figure 7 D). These pharmacological results further strengthen our conclusions from genetic approaches that functional impairment of FAO-committed metabolic reprogramming inverts AAD sensitivity of tumors growing in an adipose environment. Similar to the genetic loss of function, etomoxir and AAD combination therapy did not significantly alter vascular density compared with the AAD-alone-treated tumors. Thus, these data further support the notion of angiogenesis-independent mechanism in supporting tumor growth. Consistent with an increased anti-cancer effect, combination therapy markedly decreased tumor cell proliferation and increased cellular apoptosis ( Figures 7 C and 7F). Quantification analysis showed that combination of etomoxir and AAD radically reduced cancer cell proliferation and increased cellular apoptosis compared with monotherapy groups ( Figure 7 F). It should be emphasized that etomoxir treatment did not affect tumor inflammation ( Figure S7 ). These data support our concept of combination therapy of AADs and FAO inhibitors for effective treatment of adipose-associated cancers.

Despite regaining AAD sensitivity in steatotic livers, tumor vascular density, permeability, and perfusion were not affected in AAD-treated sh-RNA-Cpt1-CRC and sh-RNA-Cpt1-HCC cancers relative to their corresponding controls ( Figures 6 A, 6D, 6F, and 6I). These data reconciled with the fact that vascular changes were not the primary reason responsible for AAD resistance of tumor growth. Concordant with AAD sensitivity, knockdown of CPT1 markedly decreased tumor cell proliferation in AAD-treated sh-RNA-Cpt1-CRC and sh-RNA-Cpt1-HCC relative to their corresponding NIIgG-treated control cancers ( Figures 6 B, 6E, 6G, and 6J). Conversely, cellular apoptosis in AAD-treated sh-RNA-Cpt1-CRC and sh-RNA-Cpt1-HCC were markedly increased ( Figures 6 B, 6E, 6G, and 6J). Taken together, these results support the fact that FAO-committed metabolic reprogramming is responsible for AAD resistance through an angiogenesis-independent mechanism.

To further study the functional impact of the FAO-related metabolic reprogramming on tumor growth in steatotic livers, we took a small hairpin RNA (shRNA)-knockdown approach to functionally inactivate CPT1, which is the FAO-limiting enzyme. CRC and HCC cancer cells expressed Cpt1 mRNA and transfection of CRC and HCC cancer cells with shRNA-Cpt1 markedly inhibited Cpt1 expression ( Figures S6 A and S6B). Knowing effective knockdown of CPT1, we implanted these transfected cancer cells in steatotic livers. In both CRC and HCC cancer models, loss of function of CPT1 by shRNA-Cpt1 markedly reverted the anti-VEGF sensitivity ( Figures 6 A, 6C, 6F, and 6H ). Significant reduction of tumor volumes was evident in AAD-treated sh-RNA-Cpt1-CRC and sh-RNA-Cpt1-HCC relative to their respective shRNA-scrambled control-vehicle CRC and shRNA-scrambled control-vehicle HCC. These data provide compelling evidence that loss of function of FAO-limiting enzyme CPT1 reverts AAD sensitivity of steatotic liver cancers.

(D and I) Quantification of CD31 + tumor vessels (n = 9 random fields per group), extravasated 70-kDa dextran signals (n = 9 random fields per group), and perfusion of 2,000-kDa dextran (n = 9 random fields per group) in CRC (D) or HCC (I).

(B and G) Immunohistochemical analysis of Ki67 + proliferating cells and activated caspase-3 + apoptotic cells in CRC (B) or HCC (G). Arrows and arrowheads point to proliferating and apoptotic cells, respectively. Bar represents 100 μm.

(A and F) shRNA-Cpt1- and control-vehicle-transfected CRC (A) or HCC (F) tumor-bearing mice received NIIgG and anti-VEGF treatment. Upper panels: representative tumors. Arrows point to tumors in each group. Middle panels: extravasation of 70-kDa dextran (green). CD31 + blood vessels (red). Arrowheads indicate leaked dextran signals. Lower panels: perfusion of 2,000-kDa dextran (green). CD31 + blood vessels (red). Arrows indicate perfused tumor vessels. Bar represents 100 μm.

To determine the trigger that induced high expression of these gene products, we exposed CRC culture cells to hypoxia. Interestingly, a hypoxic condition of 3% oxygen markedly induced mRNA expression levels of these genes ( Figure 5 J). These findings demonstrate that hypoxia is the primary reason for induction of FFA uptake, transport, and metabolism. Given the fact that AAD induces hypoxia in adipose-associated tumors, upregulation of these fatty acid uptake-related gene products would inevitably occur in AAD-treated tumors, particularly in adipose-associated tumors.

Utilization of FFA as the lipid-dependent ATP catabolic reprogramming entails several transporters located in the cell membrane and cytoplasm (). Expression profiling demonstrated a nearly 7-fold increase of Cd36, a cell membrane transporter, in AAD-treated steatotic liver CRCs compared with NIIgG-treated control tumors ( Figure 4 C). Strikingly, a 37-fold increase of fatty acid-binding protein 1 (Fabp1), which is involved in FFA uptake, transport, and metabolism, was observed ( Figure 4 C). In addition, Fabp4 (2.3-fold), Slc27a2 (21.8-fold), and Slc27a5 (7.2-fold) were significantly increased ( Figure 4 C). High levels of expression of these gene products were further validated by real-time qPCR analysis ( Figure 5 I), which matched the expression levels of the gene microarray.

How are free fatty acids transported in membranes? Is it by proteins or by free diffusion through the lipids?.

It was plausible that the AAD-induced hypoxia depleted glucose supply from the circulation and jeopardized the Warburg effect, which caused the lipid-dependent metabolic reprogramming in cancer cells. If so, depletion of glucose in cell culture would also be able to exacerbate the FFA-induced cell proliferation through a hypoxia-independent mechanism. To test this possibility, CRC cells were stimulated with FFA in the presence of a low concentration of glucose. Under this low-glucose condition, FFA significantly stimulated cancer cell proliferation even under normoxic conditions ( Figure 5 G). Etomoxir inhibited FFA-stimulated cell proliferation in a dose-dependent manner, validating the fact that low glucose switches the Warburg effect to the lipid-dependent metabolic reprogramming to support cancer cell growth.

To further elaborate the functional impact of FAO-dependent metabolic reprogramming, cancer cells were treated with FFA in cell culture. Under normoxic conditions, FFA at various concentrations did not significantly augment cancer cell proliferation ( Figure 5 E). However, exposure of CRC cells to hypoxia markedly induced cell proliferation ( Figures 5 E and 5F). These findings further strengthen our conclusion that hypoxia was a primary trigger for activation of the FAO pathway. To study if AAD-augmented cell proliferation was CPT1 dependent, FFA-stimulated cells under hypoxia were treated with etomoxir, a known inhibitor of CPT1. Expectedly, etomoxir significantly inhibited FFA-induced cell proliferation ( Figure 5 F).

As anti-VEGF treatment induced high levels of tissue hypoxia in tumors, we pursued an in vitro experiment to study the role of hypoxia in modulation of AMPK and in a cell culture setting. Exposure of MC38 CRCs and Hepa1-6 HCCs to 3% hypoxia markedly induced phosphorylation of AMPK ( Figure 5 D). These results provide direct evidence that hypoxia alters phosphorylation of AMPK, thus reconciling with AAD-induced hypoxia in tumors. The hypoxia-induced activation of the FAO pathway was particularly enhanced in tumors grown in steatotic livers relative to those grown in healthy livers. Reconciling with our present findings, a recent work also links the causational relation between hypoxia and AMPK ().

To gain mechanistic insights, we analyzed the FAO-related crucial regulators in anti-VEGF-treated and non-immune NIIgG-treated healthy liver CRCs and steatotic liver CRCs. AAD treatment significantly increased levels of phosphorylated AMP-activated protein kinase (p-AMPK) in both healthy liver and steatotic liver CRCs ( Figures 5 A–5C ). Strikingly, tumor cells implanted in steatotic livers possessed high contents of lipid droplets relative to those implanted in healthy livers ( Figures 5 A and 5B), indicating an active lipogenic process occurring in tumor cells.

(G) MTT analysis of FFA-stimulated cell proliferation under a glucose-depleted condition in the absence and presence of various concentrations of etomoxir (n = 5–6 samples per group).

(F) MTT analysis of FFA-stimulated cell proliferation under a hypoxic condition in the absence and presence of various concentrations of etomoxir (n = 5–6 samples per group).

Genome profiling showed marked upregulation of ATP catabolism energy metabolism-related genes in anti-VEGF-treated steatotic liver CRCs but not in anti-VEGF-treated healthy liver CRCs ( Figures 4 D and 4E). The top five ATP-binding cassette (ABC) transporter-related genes were Abcb4, Abcc2, Abca6, Abcb11, and Abcd2. Consistent with gene expression profiling, ATP synthesis in anti-VEGF-treated steatotic liver CRCs remained at high levels, whereas the amount of ATP molecules in anti-VEGF-treated healthy liver CRCs was significantly reduced ( Figure 4 F). Similarly, FFA molecules were markedly reduced in anti-VEGF-treated healthy liver CRCs, whereas anti-VEGF-treated steatotic liver CRCs showed high levels of FFA ( Figure 4 F). Together, these data suggest the existence of a causational relation between lipid metabolic reprogramming and high ATP production in AAD-treated adipose-associated tumors.

The incongruity between the magnitudes of angiogenesis suppression and high rates of tumor growth in anti-VEGF-treated adipose- and fatty liver-associated tumors prompted us to investigate underlying mechanisms with a special focus on lipid metabolic reprogramming. Since lipid metabolic reprogramming in steatotic livers would likely be a candidate energy pathway contributing to continuous tumor growth in the presence of a minimal number of blood vessels, we focused our efforts on detailed studies of the FAO-committed pathway. For subsequent studies, we chose the fatty liver tumor models because of their intrinsic resistance to AAD treatment. Genome-wide expression profiling of tumors showed significant increases of FAO-related gene expression in anti-VEGF-treated steatotic liver CRCs ( Figures 4 A and 4B ). The top five upregulated FAO-related genes were Ehhadh, Acox2, and Acot112, Acad11, and Acsm3, and the top five upregulated FFA transporter-related genes were Fabp1, Slc27a2, Slc27a5, Cd36, and Fabp4 ( Figures 4 A–4C). Notably, Cpt1a, ranked number seven, was further increased after AAD treatment. Consistent with our findings, anti-VEGF-induced hypoxia has been shown by others to increase lipid transport in cancer cells (). In non-steatotic liver CRCs, these FAO- and FFA transporter-related genes remained unchanged or even downregulated ( Figures 4 A–4C). In addition, other FAO- and FFA transporter-related genes were also markedly upregulated in the anti-VEGF-treated steatotic liver CRCs but not in the anti-VEGF-treated healthy liver CRCs ( Figures 4 A–4C). These findings suggest that AAD treatment triggers a lipid metabolic reprogramming in tumors grown in adipose environments.

(D) Volcano plots of energy metabolism-related genes in CRC tumors implanted in healthy and steatotic livers. Anti-VEGF-induced top five energy metabolism-related upregulated genes in CRC tumors implanted in steatotic livers are indicated by arrows (triplicates).

(A) Volcano plots of FAO-related genes in CRC tumors implanted in healthy and steatotic livers. Anti-VEGF-induced FAO (green)- and FFA transporter (red)-related top five upregulated genes in CRC tumors implanted in steatotic livers are indicated by arrows (triplicates).

It was plausible that metabolic changes in adipocytes might also affect adjacent cancer cell metabolism. To explore this possibility, gene expression profiling of CRCs implanted in WAT and in the subcutaneous tissue was compared. Consistent with our previous findings, expression of inflammation-related genes was not significantly altered between CRCs implanted in WAT and the subcutaneous location ( Figure S5 A), suggesting that inflammation did not play a major role in our model. Moreover, genome-wide expression profiling showed several FAO-related genes, including Cpt1a, Acsl4, and Acad10, were increased in WAT-CRC ( Figures S5 B–S5D).

We next investigated the impact of anti-VEGF treatment on the TME. One of the striking findings was that tumors experienced a high magnitude of hypoxia in anti-VEGF-treated tumors grown in adipose tissues. Anti-VEGF-treated WAT-CRC, WAT-PDAC, and fatty liver-CRC tumors exhibited extremely high hypoxia relative to their corresponding tumors grown in the non-adipose subcutaneous location ( Figures 3 A–3P ). In non-adipose and non-fatty liver tumors, anti-VEGF therapy also significantly increased tissue hypoxia ( Figure 3 ), consistent with AAD-induced reduction of vascularity and blood perfusion ( Figure 2 ). Consequently, a marked decrease of tumor cell proliferation and a significant increase of cellular apoptosis were observed in non-adipose CRC and PDAC, leading to a decreased proliferation/apoptosis indexes ( Figure 3 ). Notably, despite marked decrease of vascular density and increase of tumor hypoxia, malignant cells in anti-VEGF-treated WAT-CRC, WAT-PDAC, and CRC-fatty liver tumors showed high rates of proliferation, which were indistinguishable from those of their corresponding NIIgG-treated control tumors ( Figure 3 ). Moreover, these anti-VEGF-treated adipose-associated tumors also contained a small number of apoptotic cells, resulting in high proliferation/apoptosis indexes ( Figure 3 ). Similar findings were also obtained from the steatotic liver-CRC model ( Figures 3 and S4 A). Moreover, macrophage depletion did not alter the tumor tissue hypoxia, indicating inflammation was not the cause of tissue hypoxia ( Figures 3 and S4 B–S4D). These findings suggest the possibility of the existence of angiogenesis-independent mechanism of tumor growth.

(L–P) Quantification of pimonidazole + (n = 5–8 random fields per group), CA9 + (n = 4 random fields per group), Ki67 + (n = 5–6 random fields per group), and cleaved caspase-3 + signals (n = 5–6 random fields per group) of NIIgG- and anti-VEGF-treated CRCs implanted in non-steatotic and steatotic livers.

(K) Representative micrographs of pimonidazole + , CA9 + , Ki67 + , and cleaved caspase-3 + signals of NIIgG- and anti-VEGF-treated CRCs implanted in healthy and steatotic livers. Arrows and arrowheads point to Ki67 + and cleaved caspase-3 + signals, respectively. Bar represents 100 μm.

(B–E and G–J) Quantification of pimonidazole + (n = 4–7 random fields per group), Ki67 + (n = 4–6 random fields per group), and cleaved caspase-3 + signals (n = 4–7 random fields per group) of NIIgG- and anti-VEGF-treated non-adipose and adipose CRC (B–E) and PDAC (G–J).

(A and F) Representative micrographs of pimonidazole + , Ki67 + , and cleaved caspase-3 + signals of NIIgG- and anti-VEGF-treated non-adipose and adipose CRC (A) and PDAC (F). Arrows and arrowheads point to Ki67 + and cleaved caspase-3 + signals, respectively. Bar represents 100 μm.

Unexpectedly, immunohistochemical analysis demonstrated that CRCs in steatotic livers were highly sensitive to anti-VEGF treatment, with only a minimal number of microvessels, similar to anti-VEGF-treated CRCs grown in non-steatotic control livers ( Figures 2 C and 2D). Consistently, blood perfusion and vascular permeability were markedly decreased in anti-VEGF-treated non-steatotic and steatotic CRCs compared with their respective controls ( Figures 2 C, 2D, and S3 G). Vascular perfusion was quantitatively analyzed by two independent methods. Similar findings were validated in an HCC model, showing that steatotic liver HCC tumors possessed a high magnitude of anti-VEGF drug resistance, but tumor microvessels were highly sensitive to AAD ( Figures 2 E–2H and S3 H). The findings from two clinically relevant tumor models show that tumors grown in steatotic livers are highly resistant to AAD and pose an incongruity between tumor growth and vascularization.

In healthy livers, anti-VEGF treatment markedly inhibited metastatic CRC growth ( Figures 2 A and 2B). Quantification analyses showed that liver tumor volume, liver weight, visible surface metastatic tumors, and luciferase luminescent signals were all significantly inhibited in the VEGF-treated group relative to the NIIgG-treated animals ( Figures 2 A and 2B). These findings were consistent with the therapeutic effect of anti-VEGF in non-adipose tissues. Surprisingly, CRC tumors grown in steatotic livers were highly resistant to anti-VEGF therapy. Tumor burdens in NIIgG- and VEGF-treated steatotic liver CRCs were nearly indistinguishable, exhibiting high numbers of metastatic nodules ( Figures 2 A and 2B). In contrast, tumors grown in the spleens of the same animals with steatotic livers were highly sensitive to anti-VEGF therapy ( Figure S3 C), suggesting that anti-VEGF resistance was constrained to hepatic steatosis. It was possible that HFD-induced obesity and fatty livers produce a systemic effect on tumor growth. To address this issue, tumors implanted in spleens of steatotic liver mice did not augment growth rates ( Figure S3 C). In another experiment, tumors were subcutaneously implanted in healthy mice and HFD-fed mice. No significant differences of tumor growth or inflammation were observed ( Figures S3 D and S3E). It was also possible that HFD-induced obesity and fatty livers induce extracellular matrix (ECM) change, which might affect tumor growth. However, in our experimental settings, no significant differences of tumor or liver ECM were observed ( Figure S3 F).

In our steatotic liver model, it was highly plausible that hepatic steatosis was accompanied by steatohepatitis, which would affect drug responses. To study the inflammatory response in steatotic livers, liver tissues were stained with monocyte-/macrophage-specific markers. Immunohistochemical staining of healthy and steatotic livers with F4/80 and Iba1 pan macrophage markers showed no significant differences ( Figure S3 B). Similar to CRC tumors grown in WAT and subcutaneous locations, FACS analysis of various inflammatory and immune cell populations did not reveal significant differences in CRC tumors grown in healthy and steatotic livers ( Figure S2 ).

To further validate AAD resistance of WAT-associated tumors, we performed similar experiments using fatty liver tumor models. To recapitulate the clinical situation, we developed both CRC- and HCC-steatotic liver models for our studies. In a CRC-steatotic liver model, CRC cells were implanted in the spleen, allowing cancer cells to be naturally inoculated in healthy and steatotic livers. It was possible that a high vessel density in the adipose tissue might cause an accelerated tumor growth rate relative to tumors grown in a non-adipose environment (). Furthermore, vessel cooption might provide a mechanism of AAD resistance (). The fatty liver model would eliminate this concern because microvessel density in steatotic livers was lower than that in healthy livers ( Figure S3 A). Despite the low density of microvessels, HCC and CRC exhibited an accelerated growth rate in steatotic livers ( Figures 2 A and 2E ), excluding the possible involvement of preexisting hepatic vessels in our system.

(D and H) Quantification of CD31 + tumor vessels (n = 8 random fields from three to six independent tumor samples per group), extravasated 70-kDa dextran signals (n = 5–6 random fields from three to six independent tumor samples per group), and perfusion of 2,000-kDa dextran (n = 6–8 random fields from three to six independent tumor samples per group) in CRC (D) and HCC (H) cancers.

(C and G) Micrographs of CD31 + microvessels (red), leakiness of 70-kDa dextran (green in middle panels), and perfusion of 2,000-kDa dextran (green in lower panels) in NIIgG- and anti-VEGF-treated non-steatotic and steatotic CRC (C) and HCC (G) cancers. Arrows in upper panels point to CD31 + tumor vessels. Arrowheads in middle panels indicate extravasated dextran signals. Arrows in lower panels indicate perfused tumor vessels. Bar represents 100 μm.

(B and F) Quantification of liver weight (n = 3–6 mice per group), liver tumor volume (n = 5–7 mice per group), surface visible nodules (n = 5–7 mice per group), and photon counts (n = 6–10 mice per group) in CRC (B) and HCC (F) cancers.

(A and E) Morphological and bioluminescent imaging analyses of tumor signals in healthy and steatotic livers. Arrows indicate surface CRC (A) and HCC (E) tumor nodules in healthy and steatotic livers. Bar represents 1 cm.

Surprisingly, despite resistance to anti-VEGF therapy, adipose CRC and PDAC tumor vasculatures showed high sensitivity to anti-VEGF treatment ( Figures 1 I–1L). AAD-induced microvascular reduction in adipose- and non-adipose-associated tumors was nearly indistinguishable and had no statistical significance ( Figures 1 I–1L). In non-adipose CRC and PDAC tumors, anti-VEGF treatment significantly increased vascular pericyte coverage compared with the non-immune immunoglobulin G (NIIgG)-treated control tumors ( Figures 1 I–1L). However, anti-VEGF treatment did not alter vascular pericyte coverage in white adipose tissue (WAT)-CRC and WAT-PDAC tumors. Treatments of non-adipose CRC, WAT-CRC, and -PDAC with the VEGF blockade markedly protected tumor vessels from leakage of 70-kDa dextran molecules ( Figures 1 I–1L). Surprisingly, microvasculatures in NIIgG-treated WAT-CRC and WAT-PDAC showed marked increases of blood perfusion ( Figures 1 I–1L), which might contribute to an accelerated tumor growth rate in adipose tissues. These data show that the magnitude of AAD-induced vascular suppression does not directly correlate with tumor growth rates in adipose tissues.

To validate these initial findings from the CRC model, we chose another tumor model, a pancreatic ductal adenocarcinoma (PDAC) model, which also naturally originates in an adipose environment and shows resistance to AAD treatment in human patients (). Similar to the CRC model, PDAC growing in iWAT showed intrinsic resistance to anti-VEGF treatment relative to those tumors growing in a non-adipose environment ( Figures 1 E–1H). Statistical analysis showed that anti-VEGF treatment of adipose PDAC did not result in significant inhibition of tumor growth. However, non-adipose PDAC exhibited significant inhibition to anti-VEGF therapy ( Figures 1 E–1H).

In addition to macrophages, other inflammatory cells might also participate in tumor growth and alteration of drug response. To explore this issue, we employed fluorescence-activated cell sorting (FACS) analysis using a panel of markers that discriminate different populations of inflammatory cells and immune cells in our study. CRC tumors grown in lean and HFD-fed obese mice showed no significant differences of various populations of leukocytes, including the total CD45population; CD45, B220population (B cells); CD45, CD3, CD4, CD8population (T cells); CD45, CD3, CD4population (T cells); CD45, CD3population (T cells); CD45, MHCII, CD11bpopulation (myeloid cells); CD45, MHCII, CD11b, CD11c, population (myeloid cells); CD45, NK1.1population (natural killer cells); CD45, Ly6G-Ly6C, SSCpopulation (granulocytes); and CD45, Ly6G, SSCpopulation (granulocytes) ( Figures S2 A–S2C). We also performed similar FACS analysis on CRC grown in healthy and steatotic livers. Again, no significant differences were seen between the two groups ( Figures S2 D–S2F). These results demonstrate that infiltration of inflammatory cells including various immune cells is less likely to contribute to differential drug responses.

Since macrophages in the TME often exhibit an M2 phenotypic transition, which has been associated with tumor growth and invasion, we further analyzed the CD206M2 macrophage subpopulation. The CD206M2 macrophage population was unchanged in CRC tumors grown in the non-adipose location of normal diet- and high-fat-diet (HFD)-fed mice ( Figures S1 E and S1F). These findings suggest that macrophage polarization is less likely to contribute to the differential AAD response in our experimental settings.

To further exclude the involvement of inflammatory macrophages in anti-tumor resistance, we employed an anti-CSF1R neutralizing antibody for treatment. Anti-CSF1R sufficiently depleted macrophage infiltration in tumors grown in adipose and non-adipose locations ( Figure S1 C). Despite these marked effects, anti-CSF1R treatment did not alter tumor growth rates in subcutaneous locations and iWAT ( Figure S1 D). These results provide additional supportive evidence showing that inflammatory cells are less likely responsible for the differential drug response of CRC in non-adipose and adipose locations.

To exclude the involvement of inflammatory cells in anti-tumor resistance, we treated CRC tumor-bearing mice with clodronate, a liposome-based agent to deplete macrophages. The total number of Iba1macrophages was similar in the vehicle-treated control subcutaneous and iWAT tumors ( Figure S1 A). This result demonstrates that tumors grown in the adipose environment did not augment an elevated inflammatory phenotype. Clodronate treatment effectively ablated macrophages in tumors grown in non-adipose subcutaneous and iWAT locations ( Figure S1 A). Despite ablation of inflammatory cells, the growth of vehicle- and clodronate-treated tumors showed no difference in both groups ( Figure S1 B).

To study AAD response of various tumors implanted in adipose and non-adipose environments, we chose the commonly used subcutaneous tumor implantation on the middle dorsal region as a non-adipose site and the inguinal white adipose tissue (iWAT) tumor implantation as an adipose site. A CRC model was chosen for several overt reasons: (1) CRC naturally arises from an adipose-associated environment; (2) bevacizumab is the first US Food and Drug Administration-approved AAD for the treatment of CRC in human patients; and (3) CRC patients often develop acquired AAD resistance. A previously well-characterized rabbit anti-mouse anti-VEGF neutralizing antibody was used in our study (). Surprisingly, implantation of CRC in iWAT showed significant resistance to anti-VEGF treatment relative to those tumors implanted in the subcutaneous location ( Figures 1 A–1D ). In the non-adipose location, anti-VEGF treatment resulted in approximately 74% inhibition of tumor growth, whereas in the adipose location the same CRC tumor receiving the same anti-VEGF drug only showed 21% inhibition ( Figures 1 A–1D). Thus, CRC growing in an adipose environment showed discernable resistance to anti-VEGF therapy.

(J and L) Quantification of CD31 + tumor vessels (n = 5–10 random fields per group), pericyte-associated vessels (n = 5–10 random fields per group), extravasated 70-kDa dextran signals (n = 4–7 random fields per group), and perfusion of 2,000-kDa dextran (n = 4–7 random fields per group) in CRC (J) and PDAC (L) cancers.

(I and K) Micrographs of CD31 + microvessels (red) in association with NG2 + pericytes (green in upper panels), leakiness of 70-kDa dextran (green in middle panels), and perfusion of 2000-kDa dextran (green in lower panels) in NIIgG- and anti-VEGF-treated non-adipose and adipose CRC (I) and PDAC (K) cancers. Arrows in upper panels point to NG2 + pericytes in association with tumor vessels. Arrowheads in middle panels indicate leaked dextran signals. Arrows in lower panels indicate perfused tumor vessels. Bar represents 100 μm.

(A–H) CRC (A–D) and PDAC (E–H) tumors implanted in subcutaneous (non-adipose) and inguinal WAT were treated with a NIIgG or an anti-VEGF neutralizing antibody (n = 8–10 mice per group). Tumor growth (A–C and E–G) was measured as volumes (A, B, E, and F) and weight (C and G). Percentages of tumor inhibition were calculated (D and H).

Discussion

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Hosaka K.

Nakamura M.

Cao Y. Co-option of pre-existing vascular beds in adipose tissue controls tumor growth rates and angiogenesis. VEGF inhibition causes vascular regression and tissue hypoxia in tumor tissues and their surrounding healthy adipose tissues. In this regard, tumors in steatotic livers and adjacent to adipose tissues would experience more hypoxia than non-adipose tumors. Indeed, our present experimental results support this notion, showing the existence of tissue hypoxia in adipose tumors. In both in vitro and in vivo experimental settings, we show that AAD-triggered hypoxia is the primary driving force for switching to the lipid-dependent metabolic reprogramming in tumor cells. FAO-committing crucial molecular components, including AMPK, are significantly upregulated, indicating activation of the FAO pathway in cancer cells in AAD-treated adipose tumors. Hypoxia might also trigger lipolysis in tumor adjacent adipose tissues that release more FFA. This interesting possibility warrants future investigation. In addition, hypoxia markedly increases expression levels of FFA uptake, transportation, and metabolism machineries in malignant cells to ensure the availability of FFA as a fuel for the FAO-committed energy production. Paradoxically, activation of FAO also requires oxygen for energy production and tissue hypoxia would be counterintuitive to this dogma. It is likely that mild and intermittent hypoxia induced by AAD still allows sufficient oxygen for fuel. We provide crucial evidence of the functional impact of FFA on cell proliferation under hypoxic conditions. Under the normoxic condition, FFA has no impact on cancer cell proliferation. Perhaps there is no need for cancer cell to utilize FFA for energy production because of the predominant glycolysis metabolic pathway (i.e., the Warburg effect). Under hypoxic conditions, however, both FFA uptake and metabolism pathways are activated and FFA is able to further stimulate cancer cell growth. Consistent with these in vitro findings, our recent work demonstrates that tumors in adipose tissues grow at exacerbated rates relative to non-adipose tissues (). These data also imply that the FAO-committed bioenergetic pathway also significantly contributes to tumor expansion under hypoxic conditions.

Both genetic and pharmacological loss-of-function approaches show that targeting CPT1 is an effective approach for inhibition of tumor growth in adipose environments. Importantly, a combination therapy targeting the FAO and VEGF pathways produced superior anti-cancer effects compared with monotherapies. These findings have created a concept of combination therapy consisting of AADs and FAO inhibitors, which would be particularly effective for treatment of AAD-resistant cancers growing in adipose environments.