A Different Exosome Secretion Pattern Characterizes Patient-Derived Colorectal Cancer Multicellular Spheroids and Their Mouse Xenografts

Simple Summary Exosomes have a role in tumorigenesis and metastatic dissemination, their material content and size being associated with poor prognosis of colorectal cancer (CRC). Our work aims to investigate their secretion patterns in CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and their mouse xenografts, to unveil possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. Our results show that MTSs’ exosome secretion pattern depends on their structural complexity: few-layer spheroids show a lesser exosome secretion, limited to the apical domain of cancer cells; secretion increases in multilayered spheroids and is visible from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in spheroids. The influence of the surrounding environment of non-tumor cells may account for the difference in exosome secretion patterns between spheroids and xenografts. Abstract Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes’ role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them “in situ” in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs’ exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.


Introduction
Epidemiological data on colorectal cancer (CRC) incidence and mortality (according to the World Health Organization GLOBOCAN database) show that CRC is the third most commonly diagnosed cancer in males and the second in females around the world, even if marked differences in rates exist among countries [1]. Stand-alone surgery is generally curative in 40% of patients with CRC stages 1 or 2, and 5-year survival rates reach 90% [2]. This approach is not sufficient for the management of advanced stages and metastatic CRC, which represent about 30% of cases at the time of diagnosis [3]. For this kind of patient, chemotherapy, radiotherapy, and their combination are used, but a marked heterogeneity in patients' clinical responses exists [4,5], accounting for poor survival rates [6].
One strategy to reduce mortality in CRC is, on the one hand, to succeed in finding biomarkers for early detection and, on the other, the development of personalized therapies to treat patients in the more advanced stages of the disease. The oncology community is moving towards patient-tailored cancer therapy, taking into account the unique molecular profile of each patient's cancer. This innovative approach is improving responses to therapy [7,8], and the development of up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage and genetic diversity is needed to understand some fundamental aspects of patient-specific genetic alterations, not only in CRC arising but moreover in its metastatic behavior and responsiveness to therapy. In the last two decades, MTSs cultures have been developed from CRC and other tumors and are now considered reliable preclinical in vitro models of cancer [9]. CRC MTSs consist of 3D cultures of primary cells derived from surgical specimens, reproducing patient-specific genetic expression profiles and heterogeneity [10].
At the same time, the role of exosomes as specific biomarkers in CRC prediction and screening is emerging [11]. Exosomes are nano-sized vesicles (30-120 nm), in their single membrane, express a high and cancer-specific glycosylation profile [12] and are the carrier for various lipids, proteins, DNA fragments, and several RNA species as mRNAs, microRNAs (miRNAs), long noncoding RNAs (lncRNAs) as well as small interfering RNAs (siRNAs) [13][14][15][16][17]. The potential role of exosomes in tumorigenesis and the metastatic process was demonstrated [18][19][20]. Exosomes' role in cancer progression and metastasis is that of carriers, which actively transfer bioactive molecules between cancer cells and different cell types in the nearby and distant microenvironments. The effect of such intercellular cross-talk explicates by changing multiple cellular and biological functions in recipient cells [21][22][23]. Moreover, the poor prognosis of cancer is associated with the material content and the size of the exosomes rather than the frequency of blood circulating exosomes. Exosomes are generally imaged after their recovery from blood serum by drop-casting as isolated entities, and we aim to look at them in their native environment. We focused on an exosome secretion pattern study "in situ," observed by scanning and transmission electron microscopy patient-derived MTSs and their mouse xenograft, to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models.

CSC MTSs Isolation and Culture
• Patient A 63 aged year male underwent CRC surgery for cancer removal under the standards of the ethics committee on human experimentation of the National Institute of Health (Istituto Superiore di Sanità) authorization no.CE5ISS 09/282, as reported in [24].

Animal Procedures
Animal procedures were performed according to the Italian National animal experimentation guidelines (D.L.116/92) and upon approval of the experimental protocol by the Italian Ministry of Health's Animal Experimentation Committee.

•
Xenograft extraction and treatment Palpable xenografts were extracted, and samples were then formalin-fixed and paraffinembedded. A pathologist evaluated hematoxylin and eosin-stained sections to compare xenograft histology with that of the tumor of human origin. • Embedding procedure

Results
Our morphological investigation started with the analysis of the MTSs' three-dimensional morphology by scanning electron microscopy; we then proceeded to their ultrastructural characterization; by transmission electron microscopy, highlighting aspects related to the secretion of exosomes. Finally, an ultrastructural analysis of the xenograft was performed to compare the different experimental models and highlight similarities and differences in exosome secretion. Data on the size of exosomes and MVBs were then finally statistically analyzed.

SEM Analysis of MTSs Morphology and Exosomes Secretion
Observation of the outer morphology of MTSs by scanning electron microscopy showed that their outer surface had a variable appearance. Some MTSs appeared as compact entities with smooth surfaces or sparse, shallow furrows ( Figure 1A). No exosome secretion was observed from the cells forming the outer surface. Other MTSs showed some shallow surface grooves ( Figure 1B), corresponding to the boundaries of the underlying cells; even in this case, no exosome secretion by the outermost cells was observed. Still, other MTSs exhibited on their outer surface deep and numerous grooves ( Figure 1C), with one or more cells protruding from the surface of the spheroid itself. The cells of the outermost layer exhibit blebs and microvilli, but no exosome secretion was observed.

Results
Our morphological investigation started with the analysis of the MTSs' three-dimensional morphology by scanning electron microscopy; we then proceeded to their ultrastructural characterization; by transmission electron microscopy, highlighting aspects related to the secretion of exosomes. Finally, an ultrastructural analysis of the xenograft was performed to compare the different experimental models and highlight similarities and differences in exosome secretion. Data on the size of exosomes and MVBs were then finally statistically analyzed.

SEM Analysis of MTSs Morphology and Exosomes Secretion
Observation of the outer morphology of MTSs by scanning electron microscopy showed that their outer surface had a variable appearance. Some MTSs appeared as compact entities with smooth surfaces or sparse, shallow furrows ( Figure 1A). No exosome secretion was observed from the cells forming the outer surface. Other MTSs showed some shallow surface grooves ( Figure 1B), corresponding to the boundaries of the underlying cells; even in this case, no exosome secretion by the outermost cells was observed. Still, other MTSs exhibited on their outer surface deep and numerous grooves ( Figure 1C), with one or more cells protruding from the surface of the spheroid itself. The cells of the outermost layer exhibit blebs and microvilli, but no exosome secretion was observed.

LM (Light Microscopy) and TEM Analysis of MTSs Cells Morphology and Exosomes Secretion
Observing the internal morphology of the MTSs, at first by light microscopy on semithin sections, then by transmission electron microscopy on ultrathin sections, we identified three different types of MTSs, hereafter defined as A, B, and C. MTSs of group A were characterized as bi-layered or three-layered structures, containing pseudocyst-like structures (resembling a colonic gland; Figure 2A). Cells lining the pseudocyst lumen tightly adhere to each other and project microvilli on their apical surface. In the pseudocyst lumen, no exosome secretion was visible. The cells of the outermost layers appeared loosely adhered to each other and were separated by large intercellular spaces ( Figure 2B,C) in which cells' membrane extroversions extended ( Figure 2C,D). These threadlike extroversions were sometimes short, other times were longer and convoluted, intertwining with those of adjacent cells. In the intercellular spaces, no exosome secretion was visible.

LM (Light Microscopy) and TEM Analysis of MTSs Cells Morphology and Exosomes Secretion
Observing the internal morphology of the MTSs, at first by light microscopy on semithin sections, then by transmission electron microscopy on ultrathin sections, we identified three different types of MTSs, hereafter defined as A, B, and C. MTSs of group A were characterized as bi-layered or three-layered structures, containing pseudocyst-like structures (resembling a colonic gland; Figure 2A). Cells lining the pseudocyst lumen tightly adhere to each other and project microvilli on their apical surface. In the pseudocyst lumen, no exosome secretion was visible. The cells of the outermost layers appeared loosely adhered to each other and were separated by large intercellular spaces ( Figure 2B,C) in which cells' membrane extroversions extended ( Figure 2C,D). These threadlike extroversions were sometimes short, other times were longer and convoluted, intertwining with those of adjacent cells. In the intercellular spaces, no exosome secretion was visible. Group B MTSs were structured in three to five cell layers, and the innermost cells were arranged to form a pseudocyst-like structure ( Figure 3A). Cells lining the lumen of the pseudocyst tightly adhered to each other and presented microvilli on the apical surface and secrete ( Figure 3B). Cells in the outermost layers appeared more adherent to each other than in the same cells of group A MTSs, but intercellular spaces are still present ( Figure 3C). These cells also project finger-like membrane eversions into the intercellular spaces, but we did not observe the secretion of exosomes into these spaces ( Figure 3C) or towards the external surface of the spheroid. Group B MTSs were structured in three to five cell layers, and the innermost cells were arranged to form a pseudocyst-like structure ( Figure 3A). Cells lining the lumen of the pseudocyst tightly adhered to each other and presented microvilli on the apical surface and secrete ( Figure 3B). Cells in the outermost layers appeared more adherent to each other than in the same cells of group A MTSs, but intercellular spaces are still present ( Figure 3C). These cells also project finger-like membrane eversions into the intercellular spaces, but we did not observe the secretion of exosomes into these spaces ( Figure 3C) or towards the external surface of the spheroid.  Group C MTSs have been identified as multilayered structures whose innermost ce arrange to form a pseudocyst-like structure ( Figure 4A,B). The cells bordering the lum of the pseudocyst were tightly adhered to each other, possessed microvilli on the apic surface, and secreted a large number of exosomes ( Figure 4B) and entire MVBs.
The cells of the outermost layers were also in contact with each other ( Figure 4B), fe intercellular spaces were present, and their lumen was occupied by digitiform eversio of cell membranes. In this group of MTSs, the same cells with microvilli that excrete ex somes into the lumen of the pseudocyst can also secrete exosomes from the membrane the lateral domain, and the secretion of exosomes was observed from the middle laye cells' membrane, which spills exosomes into the intercellular spaces ( Figure 4C). No ex some secretion was observed by cells' membrane of outermost layers towards the extern surface of the spheroid, i.e., directly in the culture medium. Group C MTSs have been identified as multilayered structures whose innermost cells arrange to form a pseudocyst-like structure ( Figure 4A,B). The cells bordering the lumen of the pseudocyst were tightly adhered to each other, possessed microvilli on the apical surface, and secreted a large number of exosomes ( Figure 4B) and entire MVBs.

LM and TEM Analysis of Xenograft Morphology and Exosomes Secretion
The tumor resulting from cancer-derived MTSs xenograft in immunodeficient mice (for simplicity, we will call this the xenograft) is a tissue, a more complex entity with different characteristics than MTSs, which are a 3D cultured cell mass. Simply consider the presence of necrotic and hypoxic areas, blood vessels, nerves, and fibroblasts ( Figure 5A); altogether, those factors create a different extracellular environment that is lacking in the MTSs culture system. Xenograft contains cells arranged often in pseudocysts resembling crypts and glands of the colonic tract ( Figure 5A,B) with scarce stroma in between. No goblet or enteroendocrine cells were visible as in MTSs from whom the xenograft originated or in the patient's cancer. Those pseudocysts contained cells with a columnar shape similar to enterocytes, as well as more oval cells with large oval and indented nuclei, which did not open into the gland lumen and mitotic figures. Cell nuclei were dysmorphic, with large nucleoli and heterochromatin aggregates along the inner aspect of the nuclear membrane. The cells of the outermost layers were also in contact with each other (Figure 4B), few intercellular spaces were present, and their lumen was occupied by digitiform eversions of cell membranes. In this group of MTSs, the same cells with microvilli that excrete exosomes into the lumen of the pseudocyst can also secrete exosomes from the membrane of the lateral domain, and the secretion of exosomes was observed from the middle layers cells' membrane, which spills exosomes into the intercellular spaces ( Figure 4C). No exosome secretion was observed by cells' membrane of outermost layers towards the external surface of the spheroid, i.e., directly in the culture medium.

LM and TEM Analysis of Xenograft Morphology and Exosomes Secretion
The tumor resulting from cancer-derived MTSs xenograft in immunodeficient mice (for simplicity, we will call this the xenograft) is a tissue, a more complex entity with different characteristics than MTSs, which are a 3D cultured cell mass. Simply consider the presence of necrotic and hypoxic areas, blood vessels, nerves, and fibroblasts ( Figure 5A); altogether, those factors create a different extracellular environment that is lacking in the MTSs culture system. Xenograft contains cells arranged often in pseudocysts resembling crypts and glands of the colonic tract ( Figure 5A,B) with scarce stroma in between. No goblet or enteroendocrine cells were visible as in MTSs from whom the xenograft originated or in the patient's cancer. Those pseudocysts contained cells with a columnar shape similar to enterocytes, as well as more oval cells with large oval and indented nuclei, which did not open into the gland lumen and mitotic figures. Cell nuclei were dysmorphic, with large nucleoli and heterochromatin aggregates along the inner aspect of the nuclear membrane. To study the secretion patterns of exosomes in xenografts, images obtained by transmission electron microscopy observation of the samples were analyzed. The TEM images shown in Figure 6 demonstrate the presence of secretion activity in the apical domain of cells with microvilli surrounding the lumen of the pseudocyst. This secretion activity is intense and develops homogeneously along the apical surface. The intensity of secretion is caused by the presence of numerous MVBs ( Figure 6A-C), aligned in rows perpendicular to the apical surface of the cell ( Figure 6A-C). The MVBs release their exosome content at the base of the microvilli ( Figure 6C,D), and MVBs are often entirely secreted. To study the secretion patterns of exosomes in xenografts, images obtained by transmission electron microscopy observation of the samples were analyzed. The TEM images shown in Figure 6 demonstrate the presence of secretion activity in the apical domain of cells with microvilli surrounding the lumen of the pseudocyst. This secretion activity is intense and develops homogeneously along the apical surface. The intensity of secretion is caused by the presence of numerous MVBs ( Figure 6A-C), aligned in rows perpendicular to the apical surface of the cell ( Figure 6A-C). The MVBs release their exosome content at the base of the microvilli ( Figure 6C,D), and MVBs are often entirely secreted.
Phenomena of exosome secretion were also observed from the basolateral domain of xenograft cells facing the tissue interstitium (Figure 7). In particular, cells with microvilli were observed secreting exosomes into the lumen of pseudocyst from their apical domain but also towards the tissue interstitium from the basolateral domain ( Figure 7A,B). This secretory activity is less intense than that along the apical surface. This lower intensity is caused by the fact that there are far fewer MVBs pouring their contents into the intercellular space, and these MVBs are scattered and not organized in parallel rows as they were in the apical domain. Secretory activity of exosomes was also observed from deeper cells that do not face into the lumen of the pseudocyst and that lack polarization in the apical, lateral, and basal domains ( Figure 7C,D). No entire MVB secretion was observed from the basolateral cells' domain.
shown in Figure 6 demonstrate the presence of secretion activity in the apical domain cells with microvilli surrounding the lumen of the pseudocyst. This secretion activity intense and develops homogeneously along the apical surface. The intensity of secreti is caused by the presence of numerous MVBs ( Figure 6A-C), aligned in rows perpend ular to the apical surface of the cell (Figure 6A-C). The MVBs release their exosome co tent at the base of the microvilli ( Figure 6C,D), and MVBs are often entirely secreted. lular space, and these MVBs are scattered and not organized in parallel rows as they were in the apical domain. Secretory activity of exosomes was also observed from deeper cells that do not face into the lumen of the pseudocyst and that lack polarization in the apical, lateral, and basal domains ( Figure 7C,D). No entire MVB secretion was observed from the basolateral cells' domain.   Table 1 and Figure 9.
ples, few and sparse MVBs are aligned in one horizontal row just beneath the apical p matic membrane. In xenografts, MVBs are aligned in several vertical columns perpen ular to the apical plasmatic membrane (Figure 8A,B; see also Figure 6). Few MVBs proach the basolateral membrane in MTSs, while groups of generally four or five MV are visible near the basolateral membrane in the xenografts ( Figure 8C,D).  Table 1 and Figure 9.  MBV secretion from the apical domain of spheroid cells is five times higher than ba solateral secretion. MBV secretion from the apical domain of xenograft cells is 10 time higher than basolateral secretion. MBV secretion from the apical domain of xenograft cell is massive compared to secretion from the apical domain of spheroid cells, in a ratio o 13:1. MBVs secretion from the apical domain of xenograft cells is higher than secretion from the apical domain of spheroid cells, in a ratio of 6:1.

Analysis of Exosomes and MVBs Morphology and Size by Transmission Electron Microscopy
Following the observation of MTSs' and xenografts' different secretory modalities we focused our analysis on the morphology and size of exosomes and MVBs.
Looking at the appearance of exosomes secreted from the apical domain (in both MTSs and xenograft), we noted that they presented long filaments projecting radially from the outer aspect of the membrane. These molecules partly intertwine at their initia part, proximal to the membrane, and form a "crown" around the outer surface of the ex osome. These filaments consist of glycoproteins and glycolipids of exosome membrane highlighted by the use of tannic acid in the sample preparation process for electron mi croscopy ( Figure 10A,C). Exosomes secreted from the basolateral domain ( Figure 10B,D also showed glycoproteins and glycolipids on their membrane, although in smalle amounts than in exosomes secreted from the apical domain ( Figure 10C,D). MBV secretion from the apical domain of spheroid cells is five times higher than basolateral secretion. MBV secretion from the apical domain of xenograft cells is 10 times higher than basolateral secretion. MBV secretion from the apical domain of xenograft cells is massive compared to secretion from the apical domain of spheroid cells, in a ratio of 13:1. MBVs secretion from the apical domain of xenograft cells is higher than secretion from the apical domain of spheroid cells, in a ratio of 6:1.

Analysis of Exosomes and MVBs Morphology and Size by Transmission Electron Microscopy
Following the observation of MTSs' and xenografts' different secretory modalities, we focused our analysis on the morphology and size of exosomes and MVBs.
Looking at the appearance of exosomes secreted from the apical domain (in both MTSs and xenograft), we noted that they presented long filaments projecting radially from the outer aspect of the membrane. These molecules partly intertwine at their initial part, proximal to the membrane, and form a "crown" around the outer surface of the exosome. These filaments consist of glycoproteins and glycolipids of exosome membrane, highlighted by the use of tannic acid in the sample preparation process for electron microscopy ( Figure 10A,C). Exosomes secreted from the basolateral domain ( Figure 10B,D) also showed glycoproteins and glycolipids on their membrane, although in smaller amounts than in exosomes secreted from the apical domain ( Figure 10C,D).
Exosomes secreted in basal and inflammatory conditions from human epithelial cells had a size range of 30-90 nm [33,34]. We measured the size of exosomes secreted from the different domains in the same sample (spheroid apical vs. spheroid basolateral and xenotransplant apical vs. xenotransplant basolateral), and data were statistically analyzed (Table 2, Figure 11). Exosomes secreted in basal and inflammatory conditions from human epithelial cells had a size range of 30-90 nm [33,34]. We measured the size of exosomes secreted from the different domains in the same sample (spheroid apical vs. spheroid basolateral and xenotransplant apical vs. xenotransplant basolateral), and data were statistically analyzed (Table 2, Figure 11).   the real diameter of the vesicle will correspond not to the average value of the observ diameters but will correspond to their maximum value ( Figure 11). The distribution v ues show that values in the first three columns are about two times higher concerning t last column (70-77 nm), being the value of maximum diameter (sphere equator in Figu  11E) the lesser frequent for the values in the others classes, that recur at least two times To verify if some difference exists in exosome diameter values in different samp (MTS vs. xenograft) and different secretion sites (apical vs. basolateral), an ANOVA t with Bonferroni correction was performed ( Figure 12). As can be seen from Figure 10C, the exosomes observed in a tissue sample included in resin and cross-sectioned appear not to have all the same size, as it is when vesicles extracted by centrifugation from culture medium or serum are observed at TEM by dropcasting. In the latter, the real diameter of the vesicles corresponds to the average value of the observed diameters. In our case, the correct interpretation of the real size of the vesicles must be made, taking into account that, since these are spherical dissected structures, the real diameter of the vesicle will correspond not to the average value of the observed diameters but will correspond to their maximum value ( Figure 11). The distribution values show that values in the first three columns are about two times higher concerning the last column (70-77 nm), being the value of maximum diameter (sphere equator in Figure 11E) the lesser frequent for the values in the others classes, that recur at least two times.
To verify if some difference exists in exosome diameter values in different samples (MTS vs. xenograft) and different secretion sites (apical vs. basolateral), an ANOVA test with Bonferroni correction was performed ( Figure 12).
The size of MVBs secreted from the apical domain of spheroid and xenograft was measured and compared. MVBs secreted by the spheroid basolateral domain were scarce (being present only in type C MTSs) concerning that of xenotransplant, and no comparison between these two groups was made. Results of statistical data analysis are reported above in Table 3 and Figure 13. To verify if some difference exists in exosome diameter values in different samples (MTS vs. xenograft) and different secretion sites (apical vs. basolateral), an ANOVA test with Bonferroni correction was performed ( Figure 12).   The size of MVBs secreted from the apical domain of spheroid and xenograft w measured and compared. MVBs secreted by the spheroid basolateral domain were sca (being present only in type C MTSs) concerning that of xenotransplant, and no compa son between these two groups was made. Results of statistical data analysis are repor above in Table 3 and Figure 13.  To verify if some difference exists between MVB diameter value in MTSs vs. xen grafts, a t-test was performed and results are presented in Figure 14. To verify if some difference exists between MVB diameter value in MTSs vs. xenografts, a t-test was performed and results are presented in Figure 14.
(A) MVB diameter in spheroid secretion. (B) MVB diameter in xenograft secretion. (C) The maximum diameter value is the less frequent, and the others occur at least two times (over and above the equator).
To verify if some difference exists between MVB diameter value in MTSs vs. xenografts, a t-test was performed and results are presented in Figure 14.

Discussion
Cancer stem cells are a tumor subpopulation capable of self-renewal and are crucial for survival, proliferation, drug resistance, metastasis, and tumor recurrence [35]. We recently generated a molecularly characterized biobank of colorectal CSC-enriched lines that represent a priceless resource available for in vitro studies and for the development of CSC-based murine models that faithfully reproduce the molecular and histological features of the primary tumor [10]. This primary tridimensional cell culture of tumor-derived MTSs retains the genetic heterogeneity of the original patient tumor and displays CSC's ability to dynamically switch between CSC and non-CSC states [35,36]. CRC spheroid cultures also reproduce drug sensitivity profiles of parental tumors, thus representing an excellent preclinical model to investigate the efficacy of new anticancer therapies [10,11]. While the use of MTSs for drug testing has been the object of intense studies, the biological and structural features of MTSs are less explored.
We have previously investigated the ultrastructural features of CRC MTSs, highlighting an increased presence of stem-like cells in MTSs as compared to tumor xenografts [24]. In this study, we investigated the presence and localization of exosomes in CRC MTSs and xenografts. Exosomes play a crucial role in mediating cell-to-cell communication between CSCs, non-stem cancer cells, and other cells in the tumor microenvironment (TME), regulating processes such as tumor progression, metastasis, drug resistance, EMT, and immune evasion [37]. The results of our ultrastructural study show that spheroids have exosome secretion patterns that depend on their structural complexity. Precisely, spheroids formed by a few layers of cells (Group A), with little adhesion between them, do not show exosomes production; when the number of cell layers increases and the degree of cell adhesion of the spheroid increases (Group B), secretion of exosomes into the lumen of pseudocysts is observed; until we have, in spheroids with several cell layers and a high degree of intercellular adhesion (Group C), secretion of exosomes and multivesicular bodies both from the apical domain of cells surrounding the lumen of the pseudocysts, both from the basolateral domain of the same cells and also from cells that do not face the lumen.
As reported in [38], MTSs from different CRC cell lines organize into three main types: loose, tight, and compact, and this different organization is related to different adhesion molecules expression. Loose MTSs express integrin-mediated interactions that are subsequently substituted by N-cadherin and, finally, E-cadherin interactions. In the study of [33,34,39], the different molecular profiles of apical vs. basolateral exosome secretion were demonstrated.
Our ultrastructural results (from a patient-derived CRC cell line) correlate for the first time the molecular data from the literature with the ultrastructural imaging of the different loose, tight, and compact MTS organizations, showing its relation with different exosome secretion patterns and amounts from both the apical and basolateral surfaces. This means that exosome production can be targeted in different pathways [40] to slow its progression. Lower exosome production will result in the lowering of cancer-promoting effects triggered by exosome-carried molecules.
In xenograft models (from a patient-derived MTSs xenograft), the secretion of exosomes occurs from all domains of the tumor cells and is quantitatively greater than that observed in spheroids. The massive presence of MVBs that release their contents into the lumen of the pseudocysts or in the tissue interstitium was observed.
Our ultrastructural results show for the first time the different arrangements in rows and columns (different patterns) and the difference in the amount of MBV secretion between spheroids and xenografts, xenografts being the source of a massive exosome and MVB production.
Our findings suggest that targeting the pathways of MVB formation and release could be another way to slow down cancer progression and translate our findings into clinical applications.
This difference in exosome secretion pattern and amount between MTSs and xenografts may be possibly due to the influence of surrounding non-tumor cells, as it has been shown that exosomes are key mediators of the communication between tumor cells and the tumor microenvironment [41].
Statistical analysis conducted on measurements of exosome diameter shows that exosomes secreted from the different MTSs domains have the same size (about 70 nm), which is then also the same in those produced by xenograft cells. According to [42], the size of our observed exosomes corresponds to that of a "small exosome" (Exo-S), ranging from 60 to 80 nm. Exo-S are particularly rich in Flotillin 1, flotillin 2, tweety family member 3, tetraspanin 14, and ESCRT-I subunit VPS37B. The data in [42] indicate that exosome size, in addition to their specific cargo, may influence metastatic patterning and the systemic effects of cancer.

Conclusions
Our morphological data show that structural complexity influences exosome secretion of MTSs, in both intensity and pattern, if compared with xenograft models. Our observations add new knowledge to the ultrastructural features of CRC MTSs and xenografts. Future studies may define the mechanistic basis of different exosome secretion patterns in the two model systems.