a. Tumor immunity

III.1.a.i. Cancer and tumor immunity Definition, epidemiology and biology of cancer

Cancer, also called malignant tumor, refers to a wide range of diseases affecting any part of the organism, and is defined by a rapid abnormal cell growth and a potential to spread to other parts of the body, or in other words, to metastasize^{2, 3}. There are several different types of cancers, with lung, breast and colorectal cancers being the most common. It is the second leading cause of death worldwide, although its incidence and the cancer types vary greatly by country. The global cancer incidence, mortality and prevalence (GLOBOCAN) study estimated that 18 million people had been diagnosed with cancer and about 10 million persons died from it, in 2018 (The, 2018). It is currently estimated that, in their life, 1/5 men and 1/6 women will be diagnosed with cancer, and 1/8 men and 1/10 women will die from the disease (The, 2018). Moreover, the incidence of cancer is on the rise. Indeed, by 2030, 13 million people will die each year from cancer, with 3/4 of deaths in middle- and low-income countries (The, 2018).

Cancer arises from a multi-stage process that leads to normal cell transformation into malignant cells resulting from genetic predispositions and environmental factors, including physical, chemical, and biological carcinogens (Danaei et al., 2005; Rudolph et al., 2016; The, 2018). Major risk factors include diet, lack of physical activity, and the consumption of tobacco and alcohol (The, 2018). Depending on the type/subtype of cancer, the selective contribution of genes and environment to the development of cancer differs.

Regarding the biology of cancer, specific characteristics, acquired during tumor development, constitute the hallmarks of cancer (Hanahan and Weinberg, 2011). Malignant cells sustain a proliferative signaling, escape processes that suppress their growth and resist against cell death. In addition, they allow replicative immortality, promote angiogenesis, and support invasion and metastasis. Factors underlying these mechanisms are genome instability and tumor-promoting inflammation. Lastly, cellular metabolism reprogramming and immune escape come into play

2 https://www.cancer.gov/about-cancer/understanding/what-is-cancer

3 http://www.who.int/en/news-room/fact-sheets/detail/cancer

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(Hanahan and Weinberg, 2011). This last hallmark, evading anti-tumor immunity, will be described later.

Tumors are not constituted of simple masses of malignant cells but comprise many different type of other - non-malignant – cells (Balkwill et al., 2012; Binnewies et al., 2018; Hui and Chen, 2015;

Mbeunkui and Johann, 2009). This includes infiltrating immune cells as well as recruited stromal cells, such as cancer-associated fibroblast, mesenchymal cells, or cells constituting blood and LVs (Balkwill et al., 2012; Turley et al., 2015). In addition, features including nutrient availability, pH and oxygen tension are modified compared with non-invaded organs (Lyssiotis and Kimmelman, 2017). Altogether, the interaction between tumor cells and recruited cells constitute the tumor microenvironment (TME) (Balkwill et al., 2012; Hanahan and Weinberg, 2011; Hui and Chen, 2015; Mbeunkui and Johann, 2009). The TME, which varies greatly depending on the type and stage of cancer, plays a crucial role in carcinogenesis initiation and progression (Balkwill et al., 2012; Binnewies et al., 2018; Hui and Chen, 2015; Mbeunkui and Johann, 2009).

Mouse models for cancer research

There is a wide range of mouse models for cancer research. Tumor cells can be injected/implanted in mice, leading to orthotopic or heterotopic models (Zitvogel et al., 2016). In orthotopic models, tumor cells are injected/implanted into the organ from which originated the cancer or they reach this organ through specific mechanisms when injected in the bloodstream.

In heterotopic models, the tumor cells grow in an organ that is different from the one from which the cancer originated. Genetically-engineered mice that develop tumors spontaneously or upon induction (tamoxifen Cre/LoxP system), and carcinogen-induced models also have been established (Cheon and Orsulic, 2011; Day et al., 2015; DuPage et al., 2009; Zitvogel et al., 2016).

Finally, humanized mouse models have been developed (Landgraf et al., 2018; Zitvogel et al., 2016). Each model has its own advantages and drawbacks, such as recapitulating more or less accurately specific types of human cancer, or taking more or less time for the tumor to develop.

In our study, we used heterotopic mouse models, in which tumor cells were injected subcutaneously.

Anti-tumor immunity

Several innate and adaptive immune cells, as well as numerous molecules, are implicated in the recognition and destruction of cancer cells, a phenomenon called immunosurveillance (Zitvogel et al., 2006). The theoretical and simplified cycle of anti-tumor immunity encompasses seven main steps (Fig. 12) (Chen and Mellman, 2013, 2017). Dying tumor cells release antigens that are

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uptaken by APCs, which subsequently migrate via the lymphatics to the TdLNs, where they present these antigens to T cells. T lymphocytes, including CTLs, travel through the bloodstream and infiltrate the tumor, where they recognize tumor cells. CTLs exert their cytotoxic activity, leading to further antigen release.

LN-like tertiary lymphoid structures, in which lymphoid and stromal cells accumulate, have been described in human cancer and are most of the time associated with good prognoses (Engelhard et al., 2018; Fridman et al., 2012; Goc et al., 2013; Joshi et al., 2015).

Anti-tumor immunity is influenced by environmental, host and tumor factors, from which depend the magnitude and the kinetic of the anti-tumor immune response (Chen and Mellman, 2013, 2017). The TME negatively affects the immune system by three major mechanisms:

immunoediting, immunosuppression and immunoevasion (Chen and Mellman, 2013, 2017; Dunn et al., 2004; Zitvogel et al., 2006). Mesenchymal, stromal and cancer cells play an important role in shaping the tolerogenic TME (Dunn et al., 2004; Turley et al., 2015; Zitvogel et al., 2006).

Immune cells are greatly affected by the TME and conversely, immune cells can also contribute to tolerance in the TME, including tumor-associated (TA)-neutrophils, TA-macrophages, Tregs and myeloid-derived suppressor cells (MDSCs), which secrete anti-inflammatory cytokines (Zitvogel et al., 2006).

57 Figure 12. Anti-tumor immunity cycle.

The induction of anti-tumor immunity is a self-propagating cycle, which leads to a gradual increase of immunostimulatory factors that enhance T cell responses, in principle. This cyclic process also involves immunoinhibitory factors, leading to mechanisms of immunoregulation that can prevent or limit anti-tumor immunity. The cycle comprises 7 main steps, it starts with antigen release from anti-tumor cells and ends with tumor cell killing. This scheme depicts each step, including the primary cell types that are implicated and the anatomic location.

APCs, antigen presenting cells; CTLs, cytotoxic T lymphocytes.

Adapted from Chen and Mellman, Immunity, 2013 (Chen and Mellman, 2013).

III.1.a.ii. Cancer immunotherapies