Figure S3 Flow cytometry cell sorting strategy.
4 General conclusions and future plans
The rationale behind targeting tumor angiogenesis is based on the hypothesis that tumor cells are “starved to death” when cutting their blood supply. Numerous preclinical and clinical studies have revealed a significant heterogeneous efficacy of anti-angiogenic therapies depending on the tumor type being treated. Whereas PNETs appear to be especially sensitive to this class of drugs, clinical trials in breast cancer patients largely resulted in negative results [64, 287]. In our preclinical studies, we were able to model a similar response pattern. Whereas nintedanib monotherapy increased the survival of PNET bearing Rip1Tag2 mice, nintedanib was able to delay primary tumor growth in breast cancer transplantation models (Py2T and 4T1) for only a few days. Although PNET and breast cancer models responded considerably different to nintedanib monotherapy, mechanisms of resistance might be surprisingly similar. In both tumor types, tumor regrowth (i.e. resistance) was not accompanied by revascularization and tumor cell proliferation was sustained in largely avascular tumor regions. These observations suggest a marked adaptability of tumor cells to a rapidly changing availability of oxygen and nutrients. We would like to term this capability with “metabolic plasticity”. In resistance to the anti-angiogenic TKI nintedanib, metabolic plasticity signifies the upregulation of anaerobic glycolysis. In oxygen poor situations, glycolysis serves as an important source of ATP. In rapidly proliferating cells however, glycolysis might primarily serve to provide metabolic intermediates in order to generate macromolecules. Furthermore, in hypoxia, instead of entering into the tricarboxylic acid (TCA) cycle, pyruvate is reduced to lactate in order to regenerate the cellular pool of reducing equivalents such as NAD+. Accumulating lactate has to be exported out of the cells for instance by MCT4
[420]. Indeed, we found upregulation of MCT4 in nintedanib-resistant tumors of breast cancer and Rip1Tag2 mouse models. Importantly, the exported lactate generated in hypoxic areas might not represent a simple waste product, but might be used as a fuel for oxidative metabolism by nearby normoxic areas around remaining blood vessels (i.e. metabolic symbiosis). In summary, traditional models explaining resistance to anti- angiogenic therapies are mainly based on mechanisms ensuring reoxygenation – either by revascularization or by migration to locations with higher oxygen saturation (see section 1.4, Figure 5). However, data shown in the present thesis suggest that tumor cells acquire mechanism in order to proliferate in largely avascular tumor areas. Termed metabolic plasticity, we would like to propose a novel paradigm how tumor cells sustain a reduction of the tumor vascularization by anti-angiogenic therapies (Figure 6).
Currently and in the future we are aiming to tackle this metabolic plasticity by combining nintedanib with compounds targeting the identified resistance mechanism. So far, we were able to show additive effects when combining nintedanib with 3PO or rapamycin,
both compounds inhibiting glycolysis. In addition, shRNA mediated knockdown of MCT4 resulted in a marked delay of tumor growth and resistance to nintedanib. Since in this experiment resistant tumors were composed of cells escaping shRNA mediated knockdown, we are currently working on generating Py2T cells deficient for MCT4 employing CRISPR/Cas9 technology. Furthermore, we are in contact with a pharmaceutical company to obtain a novel inhibitor of MCT4.
Figure 6. Mechanisms of resistance to anti-angiogenic therapy.
Based on our data, we suggest that the traditional concepts how tumors escape the action of anti-angiogenic therapies should be complemented by the concept of “metabolic plasticity”. Tumors resistant to nintedanib treatment displayed a remarkable adaptability allowing them proliferate despite a sustained reduction of MVD and induction of hypoxia. Tumor cells survive these harsh conditions by upregulating glycolysis. Furthermore, lactate produced by glycolytic cells, can potentially be used by cells located in normoxic areas – a mechanism termed “metabolic symbiosis”.
Importantly, the knowledge obtained in the preclinical setting by others and us should be tested in patients. Obtaining repeated biopsies from patients before and at different time points during anti-angiogenic therapy would shed light into the question, which mechanism of resistance is actually predominant in the clinical “reality”. As our laboratory is part of a European Research Council (ERC) funded consortium (“MERiC” – Mechanisms of Evasive Resistance in Cancer) aiming to unravel mechanisms of resistance to sorafenib in HCC we will have the possibility to validate our findings in preclinical mouse models of HCC and HCC patients.
Besides addressing research questions where the ultimate goal is the translatability
tool to study the impact of acute and chronic tumor hypoxia (i.e. short- and long term nintedanib treatment) on the dynamics of metabolism in tumor cells. By employing the recently established in vivo metabolic flux analysis, one might get exciting new insights into metabolic changes induced by altering the tumor microenvironment
[421].
Finally, we would like to close the circle and end with a visionary statement by Judah Folkman from 1971: “if anti-angiogenesis is not possible, or even the concept is wrong, the careful consequences may reveal something fundamental” [167]. Almost half a century later, we have seen that anti-angiogenesis is feasible. The concept is correct and provides a powerful therapeutic opportunity in certain cancer types, but clearly not in all. Nevertheless, anti-angiogenesis revealed fundamental insights into metabolic adaptations of cancer cells. Furthermore, it will provide a unique tool to study the consequences of causing acute and chronic hypoxia, and nutrient deprivation in tumors in the context of a living organism – a complexity that is impossible to model even with the most sophisticated in vitro approaches.