Nanocomposite of Agave lechuguilla natural fibers bearing silver nanoparticles formed by bio reduction with Heterotheca inuloides aqueous solution and its antibacterial properties

Concomitant with hypoxia, an increased modification and deposition of extracellular matrix (ECM), in accordance with altered transcriptome signatures, could also be observed in

sCAV1low tumors. This could be a consequence of hypoxic conditions372 _{or an independent,}

intrinsic secondary effect caused by downregulation of CAV1, given its antifibrotic role406_. Hypoxia and aggressive tumor phenotype73,269,280–283 are not only linked by fibrosis, a condition associated with therapy-resistant and metastatic breast tumors121,407_{, but also by the role} hypoxic conditions play in: (i) maintenance of the cancer stem cell (CSC) phenotype required to generate secondary tumors408_{, (ii) premetastatic niche formation}409_{, (iii) cancer cell} motility410_{, (iv) local tissue invasion, and (v) the generation of lymphatic vessels}153,371,411_{, which} constitute a common conduit for breast cancer cell dissemination and metastasis13–15. As a general process, hypoxia contributes to cancer cell escape by providing an aggressive selection pressure for resilient stem-like tumor cells that subsequently migrate away from the primary tumor46_.

In the here described in vivo experiments it is shown that stromal downregulation of CAV1 in TNBC xenografts results in lower survival rates, higher incidence of axillary lymph node metastasis and increased lung area affected by metastasis, suggesting that, in line with

previous studies331,334_{, low stromal CAV1 TNBC tumors are more aggressive. Previous studies}

support a link between loss of Caveolin-1 (CAV1) in CAFs with poor clinical outcomes in breast cancer in terms of decreased survival, early tumor recurrence, lymph node metastasis and resistance to tamoxifen89,90,331–334. Similar clinical values of decreased stromal CAV1 levels have also been found in gastric cancer412,413_{, prostate cancer}414,415 _{,and malignant}

melanoma416_{. Conversely, it has been suggested that stromal CAV1 expression is a negative}

prognostic indicator in breast cancer correlating with increased cancer dissemination101_; however, this study was focused in local tissue invasiveness via remodeling of the stromal ECM rather than the usual lymphatic metastasis of breast cancer.

Such is the importance of lymphatic dissemination of breast cancer in the clinical setting that one of the most critical steps in staging a newly diagnosed patient with breast cancer is determining if cancer has spread to the regional lymph nodes15,417_{. On this subject, since} cancer cell presence in regional mammary “sentinel” lymph nodes is considered to be a strong predictor of recurrence and survival13,15_{, specific techniques for detecting nodal involvement} and the extension of lymphatic dissemination have been developed (Sentinel lymph node

mapping)418_.

In this regard, lymphatic propagation has been described as the preferred way for breast cancer metastatic dissemination13–15. First, cancer cells derived from the primary tumor site

102 would invade the regional “sentinel” lymph nodes followed by spreading to distal nodes. At this point, nodal metastatic cells can use either lymphatic or blood vessels for subsequent

lymphogenous or hematogenous dissemination, respectively14,15,390_.

The results presented in this work suggest that initial cancer dissemination in our xenograft mouse model occurs preferentially via lymphatic vasculature, given that the first structures found affected after mastectomy, in the luciferase (data not shown) and PET-CT metastases studies, were the axillary lymph nodes. Supporting this hypothesis, further progression of the disease resulted in the invasion of the mediastinal ganglia and the lung, whose apex is right next to the subclavian vein where the lymphatic thoracic duct and the venous system communicate419_{, as observed during histological analysis.}

This lymphatic network would constitute the escape route of cancer cells from a harsh hypoxic environment that keeps aggravating as a consequence of a dysfunctional vasculature - hypoxia - proangiogenic signaling imbalance loop. In addition, the increased interstitial fluid pressure present in solid tumors, caused by vascular dysfunction and worsened by fibrosis, all of them features aggravated in sCAV1low tumors, could also be a factor facilitating tumor cell escape from the primary tumor, as in an attempt to achieve homeostasis that interstitial fluid

would be released through the lymphatic system15_.

Effective transport of cancer cells through the lymphatic system ends up entering the thoracic duct, from where these cells have access to venous blood15_{. In addition, recent studies report} an alternative hybrid route for breast cancer cell dissemination in mice via invasion of local blood vessels within a node14,420_{.Once breast cancer cells enter the blood circulation they elicit} the establishment of metastatic lesions in the lungs, liver, brain and bones. In the case of basal-

like breast cancers (BLBCs), a classification under which most TNBCs are included40,41_{, the}

preferred metastatic sites are brain, lung and distant lymph nodes, and to a significantly lesser

degree liver and bone16_{. Thus, our experimental xenograft model, using genetically modified}

CAFs to dissect stromal CAV1 function, recapitulated such natural metastatic tropism, as preferred target organs for metastasis were lung and distal lymph nodes, and with rare exceptions, the liver.

Direct hematogenous and gradual lymphogenous dissemination routes are not mutually excluding, in this regard the enhanced permeability of low stromal CAV1 tumor vasculature could also constitute a facilitating factor for tumor cell intravasation and subsequent metastasis205,229,313_.

As already mentioned, cytokines secreted by CAFs present in the tumor stroma, such as

CCL2421 _{and IL-8}368,399_{, have also been shown to increase breast cancer stem cells (CSCs)}

103 upregulation of these cytokines in CAFshCAV1 fibroblasts could also exert an intrinsic influence on the observed phenotype of increased aggressiveness in sCAV1low tumors. Different studies have attempted to explain the increased aggressiveness of sCAV1low tumors by virtue of two non-mutually-excluding models: a “Reverse Warburg effect”87 _{and the}

“autophagic tumor stroma”422 _{models, whereby loss of CAV1 in CAFs results in the production}

of metabolites and recycled products used by cancer cells. While these models explain the impact on cancer cell growth and survival, the connection to increased aggressiveness and metastasis is not so clear. Furthermore, although CAFshCAV1 fibroblasts displayed

upregulation of autophagy markers, (DRAM1, CTSB and BNIP3) (Fig. S4A), as already

described elsewhere393_{, conclusive proof of an active autophagy flux}423 _{in our CAF cellular} model was not found as both p62/SQSTM1 accumulation and LC3-II/LC3-I ratio, common active autophagy flux monitoring methods, showed no differences between CAFscr and

CAFshCAV1 fibroblasts (Fig. S4B).

Complementary to the “Reverse Warburg effect” model, the current work suggests increased hypoxic conditions in low stromal CAV1 breast tumors as: (i) a factor governing the increased aggressiveness and metastasis in these cancers and (ii) the link sustaining the positive feedback loop among dysfunctional vasculature and imbalanced proangiogenic signaling.

4.5 Endoplasmic reticulum (ER) stress: A missing link between CAV1 downregulation