Abstract and Introduction
Abstract
Background: To date, antiangiogenic therapy has failed to improve overall survival in cancer patients when used in the adjuvant setting (local-regional disease with no detectable systemic metastasis). The presence of lymph node metastases worsens prognosis, however their reliance on angiogenesis for growth has not been reported.
Methods: Here, we introduce a novel chronic lymph node window (CLNW) model to facilitate new discoveries in the growth and spread of lymph node metastases. We use the CLNW in multiple models of spontaneous lymphatic metastases in mice to study the vasculature of metastatic lymph nodes (n = 9–12). We further test our results in patient samples (n = 20 colon cancer patients; n = 20 head and neck cancer patients). Finally, we test the ability of antiangiogenic therapy to inhibit metastatic growth in the CLNW. All statistical tests were two-sided.
Results: Using the CLNW, we reveal the surprising lack of sprouting angiogenesis during metastatic growth, despite the presence of hypoxia in some lesions. Treatment with two different antiangiogenic therapies showed no effect on the growth or vascular density of lymph node metastases (day 10: untreated mean = 1.2%, 95% confidence interval [CI] = 0.7% to 1.7%; control mean = 0.7%, 95% CI = 0.1% to 1.3%; DC101 mean = 0.4%, 95% CI = 0.0% to 3.3%; sunitinib mean = 0.5%, 95% CI = 0.0% to 1.0%, analysis of variance P = .34). We confirmed these findings in clinical specimens, including the lack of reduction in blood vessel density in lymph node metastases in patients treated with bevacizumab (no bevacizumab group mean = 257 vessels/mm, 95% CI = 149 to 365 vessels/mm; bevacizumab group mean = 327 vessels/mm, 95% CI = 140 to 514 vessels/mm, P = .78).
Conclusion: We provide preclinical and clinical evidence that sprouting angiogenesis does not occur during the growth of lymph node metastases, and thus reveals a new mechanism of treatment resistance to antiangiogenic therapy in adjuvant settings. The targets of clinically approved angiogenesis inhibitors are not active during early cancer progression in the lymph node, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases.
Introduction
Although antiangiogenic therapy is standard of care for several advanced (metastatic) cancers, all phase III clinical trials of antiangiogenic therapy to date have failed in the adjuvant setting. The presence of lymph node metastases—the most common form of cancer dissemination—dictates treatment decisions, however their reliance on angiogenesis for growth has not been reported. Furthermore, observations from preclinical and clinical studies suggest that lymph node metastases and primary tumors can respond differently to the same therapeutic regimen. The clinical relevance of lymph node metastases has been the subject of debate for many years. Some argue that the presence of lymph node metastasis only demonstrates the ability of the cancer to metastasize and that disease in the lymph node is inconsequential. The strong predictive power of lymph node metastases has led others to hypothesize that cancer cells in the lymph node can exit and spread to distant metastatic sites. These advocates argue disease in lymph nodes needs to be treated in order to prevent distant metastasis and ultimately eradicate disease from the patient. Likely the answer lies in between, depending where on the spectrum of progression to distant metastasis the cancer is diagnosed. These issues highlight our fundamental lack of understanding of the biology of how metastatic cancer cells grow in a lymph node and affect the overall prognosis for the patient, limiting our ability to discover effective adjuvant therapy to treat lymph node metastases.
We and others have previously shown that antiangiogenic therapy did not stop the seeding or growth of lymph node metastases, but no mechanism of failure has been determined. Nonsprouting angiogenesis mechanisms to sustain tumor growth, such as vessel co-option and intussusception, have been implicated in the growth of lung, liver, and brain metastases and are thought to play a role in resistance to antiangiogenic therapy. Based on these findings, we hypothesized that early growth of lymph node metastases is not dependent on sprouting angiogenesis.
Although reports show reduced vascular density in lymph node metastases compared with corresponding primary tumors and surrounding normal lymph node, these data do not describe the degree of angiogenesis or whether the vessels are functional. Here, we introduce a novel model to longitudinally image the formation and growth of metastatic tumors in lymph nodes and reveal the surprising lack of sprouting angiogenesis, despite the presence of hypoxia in some lesions. Treatment with two different therapies designed to target sprouting angiogenesis showed no effect on the growth or vascular density of lymph node metastases in our models. These data are corroborated in clinical specimens and further add to mechanisms for the failure of antiangiogenic treatments in adjuvant settings.