The most frequent cancer among women worldwide is breast cancer [1]. The most aggressive and invasive kind of breast cancer, triple-negative breast cancer (TNBC), has an extremely poor prognosis [2]. Recurrence and metastasis are likely events in up to 70% of TNBC patients [3]. In breast cancer, metastasis is a significant cause of death. The primary sites of metastases in this illness are the bone, lung, and liver [4]. In a metastatic cascade, particular tumor cells separate from the initial tumors, move through the blood, lymphatic, or primo vascular system (PVS), and exit the circulation to form a new tumor in the appropriate tissue [5,6].

The PVS, a third circulatory system that matches the physical foundation of the traditional Qi channel of acupuncture, was discovered in the early 1960s. This system, which Bong-Han Kim first identified as a new circulatory system in mammals, was largely forgotten until a Seoul National University team recently uncovered it in several animal organs. The system’s size (< 3 mm) and optical transparency were likely the key factors in its slow discovery [7,8].

The PVS is currently thought of as a potent conduit for cancer cell metastasis due to its widespread distribution, high density in tumor masses, and link with the tumor microenvironment (TME). Given that primary and secondary tumors are connected directly by the PVS and cancer cells can be actively transferred through this system, its function may be crucial [6]. Long-distance cell-to-cell contact and the dissemination of malignancies from the primary location to metastatic niches would be made possible by extending the PVS [9].

The TME governs functions related to tumor survival, promotion, and metastasis. Cancer cells can spread from the initial site to a distant place through a complicated and multistep metastatic cascade when they interact with the cellular and structural components of the TME [10]. To date, no studies have been done to determine how the TME affects the PVS in breast cancer. To properly analyze the link between the PVS and the TME, we adopted the mouse model of metastatic TNBC in our research.

1. Culture of cells

The Pasteur Institute of Iran’s cell bank (C604) provided the 4T1 cell line. The cells were grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and 2% penicillin-streptomycin (both from Gibco, USA) at 37^℃ in a humidified 5% CO2 environment.

2. Induction of syngeneic animal model of breast cancer

Female BALB/c mice weighing 20 to 25 grams were obtained from the Royan Institute (Iran). At a 12-hour photoperiod, the animals were kept in cages with free access to food and water. The Shahroud University of Medical Sciences Ethics Committee approved this work (May 2021), and all animal experiments were conducted per the applicable legislation (registration number: IR.SHMU.REC.1400.029).

For the orthotopic model, 4T1 cells (1 × 105 cells in 100 μL PBS) were implanted into the mammary fat pad of a normal female BALB/c mouse.

In the heterotopic model, 4T1 cells were subcutaneously injected into the mice’s flank (or the right hind limb) (1 × 105 cells suspended in 100 μL PBS) using an insulin syringe with a 32 G needle. The mice were monitored daily for their appearance and behavior characteristics.

3. Staining dye preparation

It was stained with a 0.4% (w/v) solution of trypan blue (25900048R, CORNING Cellgro, Manassas, VA 20109, USA) in PBS. The AlamarBlue solution (ThermoFisher) was used at 0.1% (w/v) in PBS.

4. In-Vivo surgical operation and visualization of the primo vascular system

Using an intraperitoneal cocktail of anesthetics (ketamine, 25 mg/kg; xylazine, 10 mg/kg), profound anesthesia was established. The skin was retracted after a 2 cm skin incision was made up and down from the surface of the tumor tissue. Trypan blue/alamarBlue staining dye was injected into the tumor side. The area was cleaned with a 0.9% saline solution after 5 minutes, and then it was magnified 100 times with a stereomicroscope for observation.

1. Heterotopic and orthotopic mouse models of metastatic breast cancer

After 35 days of tumor development in the BALB/c mice, the heterotopic and orthotopic animal models of metastatic breast cancer were created (Fig. 1). When injected into BALB/c mice, 4T1 develops highly metastatic tumors on its own that are capable of metastasizing to other organs even as the main tumor continues to grow locally. Both heterotopic and orthotopic tumor tissues underwent H and E staining and pathological confirmation (Fig. 1).

Figure 1. A metastatic mouse model of triple-negative breast cancer (TNBC) was generated. Orthotopic (A) and Heterotopic (B) mouse models of metastatic TNBC were generated. H&E staining and pathological confirmation of tumor tissue were done in both models.

2. Staining of PVS around tumor tissue

The PVS covering the tumor tissue was too transparent to be observed using a stereomicroscope. It was only detectable with the assistance of staining dyes. Some dyes were absorbed preferentially by the PVS in the tumor tissue. In our work, we tried two dyes used in previous PVS research. We found that trypan blue and alamarBlue aided in visualizing the PVS. As shown in Fig. 2, our result indicates that, in alamarBlue staining, we have a better and more visualizable analysis of the PVS pattern and density. Neither of these dyes was removed, even with many washes.

Figure 2. Primo vascular system (PVS) staining in heterotopic (A1, A2) and orthotopic (B1, B2) tumors with both trypan blue and alamarBlue dyes. For PVS staining, 200 microliters of each dye (0.4% [w/v] solution of trypan blue and 0.1% [w/v] alamarBlue solution) were added drop by drop to the tumor site. It is worth mentioning that five minutes after adding the last drop of dye, the excess dyes were cleared from the tumor site. The dyeing process was done for each color separately.

3. PVS patterns and density in heterotopic and orthotopic tumors

In TNBC, orthotopic and heterotopic syngeneic mice are the most common models used to study the TME in preclinical research. As shown in Fig. 2, our results indicated that there are not any significant differences between the PVS patterns and density between the mammary fat pad orthotopic model and the flank heterotopic model. All analyses were conducted in triplicate, and three more samples were prepared for PVS staining in orthotopic and heterotopic mouse models. Regarding the PVS pattern in heterotopic and orthotopic models, it is important to mention that no difference was observed in the macroscopic view of the PVS in the two types of tumors as documented in images captured by a digital camera. In a more detailed interpretation, the pattern of the PVS around tumors in both models was similar in terms of three characteristics in macroscopic evaluations: covering the entire tumor surface, uniform colorability of the PVS, and absence of a specific pattern of the PVS distribution around the tumor.

The most prevalent cancer in the world is breast cancer, and the most aggressive subtype of breast cancer is TNBC [11]. The alteration of the TME is a key component of novel medicines for treating cancer patients. Targeting cancer cells in the TME is a crucial area of research because the TME, which includes stroma and cancer cells, substantially impacts clinical outcomes. The most typical model in preclinical studies to examine the TME is orthotopic and heterotopic syngeneic mice [12]. This study evaluated the PVS pattern and densities in the flank heterotopic model and the orthotopic mammary fat pad model of breast cancer. Our research demonstrates that the heterotopic tumors are bigger, but for the PVS, the pattern and density of both models are remarkably comparable.

The PVS, a third element of the circulatory system made up of very small primo-vessels (PV) and primo-nodes (PN), has lately come into existence. The PVS has been recognized in mammals on the fascia encircling tumor tissue and has also been discovered to be linked to malignancies [13]. Because these vasculatures are more numerous around tumor locations and tumor cell migration is more effective inside the PVS than in the lymphatic system, the PVS may be a newly recognized mechanism in controlling cancer growth. Still, it may also be a novel channel for cancer metastasis [14]. Here, we find that both orthotopic and heterotopic models of breast cancer have the same PVS pattern and density. In actuality, it can be said that the TME has no unique influence on the PVS pattern in tumor tissues. Independent observations of the cancer-associated PVS in heterotopic breast tumors indicated that the PVS was loosely attached to the surface of the tumor, which was grown in mouse skin by inoculation of mouse mammary carcinoma cells suspended in PBS in the right flanks subcutaneously [15].

Various studies have examined the PVS using multiple staining techniques [16]. Our study’s findings demonstrated that alamarBlue is a suitable candidate for PVS visualization. Our findings indicated that alamarBlue is a superior alternative to trypan blue for PVS staining in breast tumor tissues. However, one of the limitations of our study was the lack of additional confirmation of the PVS, which highlights the need for further studies for accurate evaluations of the PVS in metastatic breast cancer.

For the first time, our data gave significant new information about the PVS in TNBC. In a mouse model of metastatic breast cancer, the TME had little impact on the PVS pattern. Creating new anti-cancer treatments may be possible by better understanding the biological connection between the TME and the PVS.

We would like to thank the research assistant of Shahroud University of Medical Sciences and all the colleagues who helped us in this project.

This work was supported by a grant from the Shahroud University of Medical Sciences (SHMU) Grant No 99132.

Mohammad Kamalabadi Farahani, Conceptualization, Methodology, Performing the Experiments (Animal Study, Cell Culture), Supervised the Experimentators, Writing- Original draft preparation; Amir Atashi Performing the Experiments (Animal Study), Writing- Reviewing and Editing; Nariman Rezaei Kolarijani, Performing the Experiments (Animal Study).

This study was approved by the Ethics Committee of Shahroud University of Medical Sciences (May 2021) (registration number: IR.SHMU.REC.1400.029).

The authors declare no conflict of interest.