Infantesa i joventut: plantació i reg de la llavor

Cancer and stromal cells face hypoxic and nutrient deficiency conditions during tumor growth. The ER, as a cellular sensor of metabolic imbalance, activates a stress response as an initial adaptation to these conditions244–247,376,431,432. Based on our evidence, ER stress activation could provide a new explanation for a self-sustained circuit leading to CAV1 downregulation in CAFs, a question that remained unsolved128,323_.

In this model, ER stress activation in CAFs, as a result of CAV1 downregulation promotes an exacerbated proangiogenic signaling that results in the formation of an impaired vascular

network accompanied by the consequent hypoxia (Fig. 34A). These increased hypoxic

conditions do not only further aggravate the proangiogenic and ER stress imbalances, they are proposed as the reason behind increased aggressiveness observed in sCAV1low tumors

(Fig. 34B).

The imbalance between pro- and anti-angiogenic cues found in tumors, a condition that the

present study shows to be aggravated in sCAV1low TNBC xenografts (Fig. 19), is responsible

for the formation of structurally aberrant and dysfunctional tumor vascular networks155_{(Fig. 18,}

20 and 21). This situation, in turn, aggravates tumor hypoxia152 _{and promotes a general} increase in aggressiveness46,73,282–284, traits also displayed by the described sCAV1low tumors

(Fig. 22, 23, 32 and 33).

A recent concept in antiangiogenic therapy is that of vascular normalization146,285_{, where} instead of aiming at a complete elimination of the tumor vasculature (vascular regression), treatments intent to restore the balance among pro- and anti-angiogenic signals in an attempt to recover blood vessel functionality266,286_{. Although this approach of improving tumor irrigation} might sounds counterintuitive, it has been shown to reduce aggressiveness and improve drug delivery.

In the scenario presented in this work, the balance in low stromal CAV1 tumors is tilted to the side of proangiogenic factor production. Among this pool of proangiogenic factors, IL-8 was

shown to be highly enriched, both in low stromal CAV1 xenograft lysates (Fig. 19B and 19D)

and CAFshCAV1 fibroblasts (Fig. 25A, B, and D). In an attempt to normalize and restore vascular functionality in low stromal CAV1 tumors, and thus observe a potential decrease in

the aggressive tumor phenotype showed by these tumors, this work proposes administration

of IL-8 inhibitors to mice during primary tumor growth. As already mentioned, one could anticipate that IL-8 inhibition in sCAV1high xenografts, where IL-8 expression levels are not upregulated, could provoke a reduction in tumor blood vessel functionality leading to an increased metastatic behavior.

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Figure 34. Proposed model for increased aggressiveness in low stromal CAV1 (sCAV1low) xenografts (A) Upon CAV1 downregulation in CAFshCAV1 fibroblasts, an aberrant proangiogenic signaling ensues derived from the activated ER stress. The imbalance in the production of pro- and anti- angiogenic cues, derived from these CAFshCAV1 fibroblasts present in xenografts (low stromal CAV1 xenografts), leads to the formation of an aberrantly dense and dysfunctional tumor vasculature. This blood vessel dysfunctionality results in increased intratumoral hypoxia, which promotes fibrosis and tumor aggressiveness. Insets of CAFs depict CAV1 in the membrane, mitochondria and the ER in blue. Red/green spheres represent pro- and anti-angiogenic cues (respectively). Darkest red spheres represent IL-8. (B) CAV1 downregulation and induction of ER stress in CAFs constitutes a positive feedback loop that is responsible for activating an exacerbated production of proangiogenic factors. This imbalance, in turn, primes the formation of an impaired tumor vasculature that promotes intratumoral hypoxia and derives in increased tumor aggressiveness. Hypoxia then constitutes a self-sustaining factor via further unbalancing pro- and anti-angiogenic cues and maintaining the ER stress induction – CAV1 downregulation loop.

These anticipated results concerning IL-8 inhibition lead to two important observations: A first one concerning the concept of vascular regression versus vascular normalization in antiangiogenic therapy, whereby either too strong an inhibition, or the inhibition (and thus dysregulation) of an otherwise normal (non-upregulated) proangiogenic factor, results in

decreased tumor vasculature functionality and a more aggressive tumor phenotype264–268, as

proposed for sCAV1high tumors. This study advocates for the latter, where a restoration of the imbalance presented by the cues governing angiogenesis in low stromal CAV1 xenografts could result in vascular normalization and a less belligerent tumor.

110 Such differential effects of IL-8 inhibition in high or low stromal CAV1 tumors leads to a second observation, bringing to the spotlight the importance of correctly stratifying patients in accordance to well-defined predictive markers for treatment response. The present study propones low CAV1 stromal and high IL-8 tumor expression levels as: (i) markers for poor prognosis of breast cancer (TNBC in this case), as a growing body of evidence has already reported89,90,331–334,391,392, and (ii) possible predictive markers for treatment response to IL-8 inhibition. Still, standardized techniques for stromal CAV1 detection in patient biopsies and further animal studies will be needed to validate the proposed theoretical predictive value before translating it into a clinical setting.

From a therapeutic point of view, normalizing aberrant angiogenesis in cancer needs new avenues of exploration given the relative inefficacy of present anti-angiogenic therapies which largely depend on ligand neutralization and perturbation of classic angiogenic signaling pathways182,266,267_{. In this regard, this newly proposed ER stress - IL-8 axis in low stromal CAV1} TNBC tumors presents itself as a potential target for ameliorating the delivery of chemotherapeutic drugs and immune cells to tumor masses through vessel normalization, and for curbing stress-induced aggressive behavior leading to metastasis. Moreover, ER stress signaling itself is amenable for pharmacological intervention- combinatorial approaches may be devised additionally including this novel opportunity for fine-tuning tumor biology and architecture.

Finally, and in a more general sense, this study, in line with a recent tendency45,46,183_{, suggests} that modulating the extracellular environment that surrounds and infiltrates tumors may provide an additional opportunity for therapeutic intervention in hard to treat cancers such as TNBC.

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