**4. Suggested evaluation parameters**

**Angiogenesis:** Angiogenesis is a physiological process that refers to new blood vessel formation. In the tumor microenvironment, angiogenesis is over-induced to sustain tumor growth and metastasis. The vascular endothelial factor is one of the parameters used to evaluate angiogenesis. This factor induces angiogenesis, and it is increased in tumor and tumor-adjacent stroma tissue and correlates with tumor aggressiveness and with the patient prognosis [66]. Another parameter for angiogenesis evaluation is the tissue hemoglobin content [56, 57]. Angiogenesis can be measured in the tumor and adjacent tissue (skin from the air pouch), with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry, ELISA, and RT-PCR.

with good prognosis of patients with different types of cancer [69, 70]. IFN-γ levels can be measured in the tumor tissue or tumor tissue supernatant with the following techniques: flow

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**Lipid rafts:** Cholesterol and sphingolipids form specific domains termed lipid rafts that regulate receptor-ligand interactions. In cancer cells, signaling protein and pro-oncogenic receptor activation correlates with their location inside the lipid rafts; disruption of lipid rafts induces apoptosis in cancer cell lines. Lipid rafts are characterized by the presence of glycosylphosphatidylinositol (GPI)-anchored proteins [71]. Also, acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), ATP citrate lyase (ACLY), and other lipogenic enzymes that promote cholesterol synthesis are altered in most tumors [72]. Lipid rafts can be measured in the tumor and adjacent tissue (skin from the air pouch), with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry,

**Liver toxicity:** Synthetic and biological compounds are often metabolized and excreted by the liver. Certain drugs can be metabolized to reactive compounds that bind to intracellular proteins inducing oxidative stress and cell death. Liver toxicity must be evaluated in early phase or preclinical studies of any drug and can be assessed by transcriptomics, cellular respiration, ATP (adenosine triphosphate), ROS (reactive oxygen species), covalent binding, apoptosis or necrosis, and bile salt export pump inhibition tests [73]. Liver toxicity can be evaluated in the liver tissue by immunohistochemistry or Western blot or in peripheral blood by flow cytometry or ELISA. Furthermore, blood tests can be included (glutamic oxaloacetic transaminase,

**Mesenchymal stem cells (MSCs):** Mesenchymal stem cells are multipotent stem cells characterized by CD73, CD105, and CD90 surface markers. MSCs have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes and are recruited to injured tissues for healing.

and CCL5, therefore promoting cancer stemness and metastasis; furthermore, they have been shown to regulate cancer cell metabolism by exosome secretion [74]. MSCs can be evaluated in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy,

**Tumor-associated macrophages:** Macrophages are specialized phagocytic cells of the immune system. In response to various stimuli, macrophages shift their phenotype to M1 or M2. M1 macrophages are associated to an inflammatory response with antitumor properties; on the other hand, M2 macrophages promote tumor growth and have anti-inflammatory properties. Tumor-associated macrophages (TAMs) found in the TME resemble M2 macrophages and correlate with poor prognosis. TAMs produce IL-23, IL-17, IL-6, PGE2, IL-10, and indoleamine 2,3-dioxygenase and CCL17, CCL18, and CCL22, which are chemotactic factors for regulatory T cells. TAMs are characterized by CD163, CD204, or CD206 surface markers [74, 75]. TAMs can be evaluated in the tumor tissue (skin from the air pouch), with the following techniques: flow cytometry, fluorescence microscopy, Western blot, and

, IL-6, IL-10, IL-17b, EGF,

cytometry, ELISA, and RT-PCR.

glutamic pyruvic transaminase, alkaline phosphatase).

Western blot, and immunohistochemistry.

immunohistochemistry.

However, under different stimuli in the TME, MSCs secrete PGE<sup>2</sup>

and ELISA.

**Cancer-associated fibroblasts (CAFs):** Fibroblasts are the most abundant cells of the connective tissue; they produce collagen for the extracellular matrix and are involved in wound healing. In the tumor microenvironment (TME), cancer cells and other stroma cells induce fibroblasts to produce tumor-promoting substances such as epidermal and hepatocyte growth factors; chemokines CXCL12, CXCL14, and CCL5 [that attract immature and suppressor immune cells]; vascular endothelial growth factor; and interleukin-6, among others. This type of fibroblasts is termed activated or cancer-associated fibroblasts (CAFs), and they correlate with tumor growth, progression, metastasis, and chemoresistance [67].

Several cell markers are used to detect CAFs, including fibroblast activation protein α, podoplanin-a, S100A4, vimentin, fibroblast-specific protein-1, platelet-derived growth factor receptors α and β, and insulin-like growth factor-binding protein [67]. CAFs can be measured in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry, ELISA, and RT-PCR.

**Indoleamine 2,3-dioxygenase 1 (IDO1):** It is a cytosolic enzyme that catabolizes tryptophan into kynurenine, a metabolite with immunosuppressive properties. IDO1 is overexpressed in more than 50% of all tumors. Increased levels of IDO1 correlate with the decrease of natural killers and specific effector T cells and increase of regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells. IDO1 also correlates with tumor progression and multidrug resistance. It is therefore considered a tumor progression biomarker and a promising therapeutic target [68]. IDO levels can be measured in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry, ELISA, and RT-PCR.

**Interferon gamma (IFN-γ):** It is a cytokine produced by natural killer cells, natural killer T cells, antigen-specific CD4 Th1 and CD8 cytotoxic effector lymphocytes, non-cytotoxic innate lymphoid cells, and mucosal epithelial cells. IFN-γ induces class I and II major histocompatibility complex expression on antigen-presenting cells, promotes natural killer activity, increases antigen presentation and lysosome activity of macrophages, activates inducible nitric oxide synthase, induces production of IgG by plasma cells, promotes adhesion required for leukocyte migration, and has direct antiviral effect (by induction of tripartite motif-containing protein 5 and apolipoprotein B-mRNA editing enzyme, among others) [69]. IFN-γ levels correlate with good prognosis of patients with different types of cancer [69, 70]. IFN-γ levels can be measured in the tumor tissue or tumor tissue supernatant with the following techniques: flow cytometry, ELISA, and RT-PCR.

**4. Suggested evaluation parameters**

ELISA, and RT-PCR.

184 Cell Culture

ELISA, and RT-PCR.

**Angiogenesis:** Angiogenesis is a physiological process that refers to new blood vessel formation. In the tumor microenvironment, angiogenesis is over-induced to sustain tumor growth and metastasis. The vascular endothelial factor is one of the parameters used to evaluate angiogenesis. This factor induces angiogenesis, and it is increased in tumor and tumor-adjacent stroma tissue and correlates with tumor aggressiveness and with the patient prognosis [66]. Another parameter for angiogenesis evaluation is the tissue hemoglobin content [56, 57]. Angiogenesis can be measured in the tumor and adjacent tissue (skin from the air pouch), with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry,

**Cancer-associated fibroblasts (CAFs):** Fibroblasts are the most abundant cells of the connective tissue; they produce collagen for the extracellular matrix and are involved in wound healing. In the tumor microenvironment (TME), cancer cells and other stroma cells induce fibroblasts to produce tumor-promoting substances such as epidermal and hepatocyte growth factors; chemokines CXCL12, CXCL14, and CCL5 [that attract immature and suppressor immune cells]; vascular endothelial growth factor; and interleukin-6, among others. This type of fibroblasts is termed activated or cancer-associated fibroblasts (CAFs), and they

Several cell markers are used to detect CAFs, including fibroblast activation protein α, podoplanin-a, S100A4, vimentin, fibroblast-specific protein-1, platelet-derived growth factor receptors α and β, and insulin-like growth factor-binding protein [67]. CAFs can be measured in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy,

**Indoleamine 2,3-dioxygenase 1 (IDO1):** It is a cytosolic enzyme that catabolizes tryptophan into kynurenine, a metabolite with immunosuppressive properties. IDO1 is overexpressed in more than 50% of all tumors. Increased levels of IDO1 correlate with the decrease of natural killers and specific effector T cells and increase of regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells. IDO1 also correlates with tumor progression and multidrug resistance. It is therefore considered a tumor progression biomarker and a promising therapeutic target [68]. IDO levels can be measured in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry,

**Interferon gamma (IFN-γ):** It is a cytokine produced by natural killer cells, natural killer T cells, antigen-specific CD4 Th1 and CD8 cytotoxic effector lymphocytes, non-cytotoxic innate lymphoid cells, and mucosal epithelial cells. IFN-γ induces class I and II major histocompatibility complex expression on antigen-presenting cells, promotes natural killer activity, increases antigen presentation and lysosome activity of macrophages, activates inducible nitric oxide synthase, induces production of IgG by plasma cells, promotes adhesion required for leukocyte migration, and has direct antiviral effect (by induction of tripartite motif-containing protein 5 and apolipoprotein B-mRNA editing enzyme, among others) [69]. IFN-γ levels correlate

correlate with tumor growth, progression, metastasis, and chemoresistance [67].

Western blot, immunohistochemistry, ELISA, and RT-PCR.

**Lipid rafts:** Cholesterol and sphingolipids form specific domains termed lipid rafts that regulate receptor-ligand interactions. In cancer cells, signaling protein and pro-oncogenic receptor activation correlates with their location inside the lipid rafts; disruption of lipid rafts induces apoptosis in cancer cell lines. Lipid rafts are characterized by the presence of glycosylphosphatidylinositol (GPI)-anchored proteins [71]. Also, acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), ATP citrate lyase (ACLY), and other lipogenic enzymes that promote cholesterol synthesis are altered in most tumors [72]. Lipid rafts can be measured in the tumor and adjacent tissue (skin from the air pouch), with the following techniques: flow cytometry, fluorescence microscopy, Western blot, immunohistochemistry, and ELISA.

**Liver toxicity:** Synthetic and biological compounds are often metabolized and excreted by the liver. Certain drugs can be metabolized to reactive compounds that bind to intracellular proteins inducing oxidative stress and cell death. Liver toxicity must be evaluated in early phase or preclinical studies of any drug and can be assessed by transcriptomics, cellular respiration, ATP (adenosine triphosphate), ROS (reactive oxygen species), covalent binding, apoptosis or necrosis, and bile salt export pump inhibition tests [73]. Liver toxicity can be evaluated in the liver tissue by immunohistochemistry or Western blot or in peripheral blood by flow cytometry or ELISA. Furthermore, blood tests can be included (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase).

**Mesenchymal stem cells (MSCs):** Mesenchymal stem cells are multipotent stem cells characterized by CD73, CD105, and CD90 surface markers. MSCs have the potential to differentiate into osteoblasts, chondrocytes, and adipocytes and are recruited to injured tissues for healing. However, under different stimuli in the TME, MSCs secrete PGE<sup>2</sup> , IL-6, IL-10, IL-17b, EGF, and CCL5, therefore promoting cancer stemness and metastasis; furthermore, they have been shown to regulate cancer cell metabolism by exosome secretion [74]. MSCs can be evaluated in the tumor tissue with the following techniques: flow cytometry, fluorescence microscopy, Western blot, and immunohistochemistry.

**Tumor-associated macrophages:** Macrophages are specialized phagocytic cells of the immune system. In response to various stimuli, macrophages shift their phenotype to M1 or M2. M1 macrophages are associated to an inflammatory response with antitumor properties; on the other hand, M2 macrophages promote tumor growth and have anti-inflammatory properties. Tumor-associated macrophages (TAMs) found in the TME resemble M2 macrophages and correlate with poor prognosis. TAMs produce IL-23, IL-17, IL-6, PGE2, IL-10, and indoleamine 2,3-dioxygenase and CCL17, CCL18, and CCL22, which are chemotactic factors for regulatory T cells. TAMs are characterized by CD163, CD204, or CD206 surface markers [74, 75]. TAMs can be evaluated in the tumor tissue (skin from the air pouch), with the following techniques: flow cytometry, fluorescence microscopy, Western blot, and immunohistochemistry.

**Tumor growth rate:** Tumor size is defined by the Response Evaluation Criteria in Solid Tumors (RECIST) as the sum of the longest diameters of the tumor mass; to report the tumor growth rate, tumor size can be measured during relevant therapy time points, for example, before treatment, after the first treatment cycle, after the last cycle of treatment, and after discontinuation of the treatment [76]. Also, the Ki67 levels, a protein in all phases of the cell cycle (G1, S, G2, and mitosis), except for the resting phase (G0), often correlate with tumor growth and progression [77]. Tumor growth can be measured with a caliper (volume), or weighted after removal, or determination of Ki67 levels in the tumor tissue by flow cytometry, fluorescence microscopy, Western blot, and immunohistochemistry.

ACC acetyl-CoA carboxylase

ATP adenosine triphosphate BrdU 5-Bromo-2′-deoxyuridine

CAFs cancer-associated fibroblasts

DMBA Dalton's lymphoma-associated antigen ELISA enzyme-linked immunosorbent assay

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ACLY ATP citrate lyase

CP cyclophosphamide

ePTFE polytetrafluoroethylene

GPI glycosylphosphatidylinositol

LDPE low-density polyethylene

PMA phorbol myristate acetate

ROS reactive oxygen species

RECIST response evaluation criteria in solid tumors

RT-PCR real-time polymerase chain reaction

Moisés Armides Franco-Molina\*, Silvia Elena Santana-Krímskaya and

Autonomous University of Nuevo León, San Nicolás de los Garza, México

Laboratory of Immunology and Virology of the Faculty of Biological Sciences of the

[1] Breslin S, O'Driscoll L. Three-dimensional cell culture: The missing link in drug discovery. Drug Discovery Today. 2013;**18**:240-249. DOI: 10.1016/j.drudis.2012.10.003

TAMs tumor-associated macrophages

TME tumor microenvironment

\*Address all correspondence to: moyfranco@gmail.com

**Author details**

**References**

Cristina Rodríguez-Padilla

FASN fatty acid synthase

MMC mitomycin C

PLLA poly(L-lactic acid)
