察爾斯大夫 美麗殿堂 Dr. Charles Meridien Palace

Cancer stem cells（CSCs，癌症幹細胞）and Stemline therapy or CSC-targeted cancer therapy

Cancer stem cells（CSCs）are cancer cells found within tumors or hematological cancers that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample.

CSCs are therefore tumorigenic（tumor-forming）, perhaps in contrast to other non-tumorigenic cancer cells.

CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease.

Existing cancer treatments have mostly been developed based on animal models, where therapies able to promote tumor shrinkage were deemed effective. However, animals could not provide a complete model of human disease. In particular, in mice, whose life spans do not exceed two years, tumor relapse is exceptionally difficult to study.

The efficacy of cancer treatments is, in the initial stages of testing, often measured by the ablation fraction of tumor mass（fractional kill）. As CSCs would form a very small proportion of the tumor, this may not necessarily select for drugs that act specifically on the stem cells. The theory suggests that Conventional chemotherapies kill Differentiated or Differentiating cells, which form the bulk of the tumor but are unable to generate new cells. A population of CSCs, which gave rise to it, could remain untouched and cause a relapse of the disease.

癌症幹細胞（Cancer Stem Cell，CSC），又稱癌幹細胞、腫瘤幹細胞，是指具有幹細胞（Stem cell）性質的癌細胞，也就是具有「自我複製」（self-renewal）以及「具有多細胞分化」（differentiation）等能力。通常這類的細胞被認為有形成腫瘤，發展成癌症的潛力，特別是隨著癌症轉移出去後，產生新型癌症的來源。

腫瘤內許多的細胞，其實只有一群細胞具有永生不死、持續分裂、分化的能力，而這些細胞，就稱為癌症幹細胞。

癌症幹細胞被認為是造成癌症轉移、復發，或是腫瘤對於化療、放射性療法產生抗性的原因之一。目前針對這個理論，學者希望研究出針對癌症幹細胞的療法，能夠專一性地殺死癌症幹細胞，以降低腫瘤產生抗藥性或是轉移的現象。

目前的癌症療法都是基於動物實驗的結果，目前的認知是只要癌症療法能將動物身上的腫瘤有效地縮小，就會被認為有其療效，然而要將實驗結果對照到人體醫療上仍有相當大的差距。比如說，以小鼠為例，生活週期很難超過兩年，如此短的生活週期無法提供癌症復發的動物模型可供研究。

而癌症幹細胞的理論認為，因為現行的治療都無法專一性的針對癌症幹細胞，而癌症幹細胞在腫瘤裡其實只佔一小部分，只要化療、放射性療法沒有殺死所有的癌症幹細胞，就會有抗藥性或是復發的風險出現。

Adoptive cell transfer

Adoptive cell transfer can refer to either the transfer of cells, most commonly immune-derived cells, back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host.

If possible, use of autologous cells helps the recipient by minimizing GVHD issues. For isolation of immune cells for adpotive transfer, a phlebotomist draws blood into tubes containing anticoagulant and the PBM（buffy coat）cells are isolated, typically by density barrier centrifugation. In T cell-based therapies, these cells are expanded in vitro using cell culture methods relying heavily on the Immunomodulatory action of Interleukin-2 and returned to the patient in large numbers intravenously in an activated state. Anti-CD3 antibody is commonly used to promote the proliferation of T cells in culture. Research into interleukin-21 suggests it may also play an important role in enhancing the efficacy of T cell based therapies prepared in vitro.

An emerging treatment modality for various diseases is the transfer of stem cells to achieve therapeutic effect. Clinically, this approach has been exploited to transfer either Immune-promoting or Tolerogenic cells（often lymphocytes）to patients to either enhance immunity against viruses and cancer or to promote tolerance in the setting of autoimmune disease, such as Type I diabetes or rheumatoid arthritis.

Cells used in adoptive therapy may be genetically modified using recombinant DNA technology to achieve any number of goals. One example of this in the case of T cell adoptive therapy is the addition of Chimeric antigen receptors, or CARs, to redirect the specificity of cytotoxic and helper T cells.

Adoptive T Cell Therapy

Adoptive T cell therapy involves the isolation and ex vivo expansion of tumor specific T cells to achieve greater number of T cells than what could be obtained by vaccination alone.

The tumor specific T cells are then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor via T cells which can attack and kill cancer.

There are many forms of adoptive T cell therapy being used for cancer treatment; culturing tumor infiltrating lymphocytes or TIL, isolating and expanding one particular T cell or clone, and even using T cells that have been engineered to potently recognize and attack tumors.

One approach has been to utilize T cells taken directly from the patient’s blood after they have received a cancer vaccine.

A unique aspect to adoptive T cell therapy is the use of tumor specific CD4+ Th1 cells which may enhance anti-tumor efficacy.

Adoptive T cell therapy strategies have largely focused on the infusion of tumor antigen specific cytotoxic T cells（CTL）which can directly kill tumor cells. However, CD4+ Th cells have a broader functionality. Th can activate antigen-specific effector cells and recruit cells of the innate immune system such as macrophages and dendritic cells to assist in antigen presentation（APC）. Moreover, antigen primed Th cells can directly activate tumor antigen-specific CTL. In addition to direct contact, Th can activate CTL through cytokines such as IL-2 which stimulate the growth and expansion of effector T cells. In addition, Th1 induce the production of opsonizing antibodies that enhance the uptake of tumor cells into APC. These activated APC can then directly present tumor antigens to T cells.

As a direct result of activating APC, antigen specific Th1 have been implicated as the initiators of epitope or determinant spreading which is a broadening of immunity to other antigens in the tumor. The phenomenon of epitope spreading has been linked with a survival benefit after immunotherapy in patients with melanoma and breast cancer. The ability to elicit epitope spreading broadens the immune response to many potential antigens in the tumor and presumably would result in more efficient tumor cell kill due to the ability to mount a heterogeneic response. In this way, adoptive T cell therapy can used to stimulate endogenous immunity.

Granzymes：Serine proteases，顆粒溶解酶

Granzymes are Serine proteases that are released by Cytoplasmic granules within Cytotoxic T cells and Natural killer cells.

Their purpose is to induce apoptosis within virus-infected cells, thus destroying them.

Granzymes when in the host cell are contained in cytotoxic granules to prevent harm to the host cell. Other locations that granzymes can be detected are in the rough endoplasmic reticulum, Golgi complex, and the trans-Golgi reticulum. The goal of the granules and perforins is to create a path way for the granzymes to follow and enter the target cells cytosol.

Granzymes are identified as being part of the serine esterase family.

Cytotoxic T cells and Natural killer cells release a protein called Perforin, which attacks the target cells. Researchers used to think that perforin creates pores within the cell membranes, through which the granzymes can enter, inducing apoptosis. However, new evidence indicates that a multimeric complex（Granzyme B, Perforin, and granulysin）can enter a cell through the Mannose 6-phosphate receptor or another receptor found in tumor cells and is enclosed in a vesicle or a sac. Perforin then allows GrB to pass through the vesicle surface and into the cell, causing apoptosis by various pathways.

They do so by cleaving caspases, especially Caspase-3, which in turn activates Caspase-activated DNase. This enzyme degrades DNA, thus inducing apoptotic cascades. Also, GrB cleaves the protein Bid, which recruits the protein Bax and Bak to change the membrane permeability of the mitochondria, causing the release of cytochrome c（which is one of the parts needed to activate caspase-9 via the Apoptosome）, Smac/Diablo and Omi/HtrA2（which suppress the Inhibitor of apoptosis proteins，IAPs）, among other proteins. As well, GrB is shown to cleave many of the chemicals responsible for apoptosis without the aid of caspase, as proven by experiments on caspase knockout mice CTL cells incubated with other cells.

In 1986 Jürg Tschopp and his group published a paper on their discovery of granzymes. In the paper they discussed how they purified, characterized and discovered a variety of granzymes found within cytolytic granules that were carried by cytotoxic T lymphocytes and natural killer cells. Jürg was able to identify 8 different granzymes and discovered partial amino acid sequences for each. The molecules were unofficially named Grs for five years before Jürg and his team came up with the name granzymes which was widely accepted by the scientific community.

Granzyme secretion can be detected and measured using Western Blot or ELISA techniques. Granzyme secreting cells can be identified and quantified by flow cytometry or ELISPOT. Alternatively, granzyme activity can be assayed by virtue of their protease activity.

Perforin-1：a protein encoded by the PRF1 gene in humans

Perforin is a cytolytic protein found in the granules of Cytotoxic T lymphocytes（CTLs）and NK cells.

Epitope：Antigenic determinant抗原決定位、抗原表位

Epitope, also known as Antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a Paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.

The epitopes of protein antigens are divided into two categories, Conformational epitopes and Linear epitopes, based on their structure and interaction with the paratope. A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. The proportion of epitopes that are conformational is unknown.

By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen.

Epitope抗原決定位、抗原表位：是指抗原表面上決定抗原特異性的化學基團。

抗原表位可被免疫系統（尤其是抗體、B細胞或者T細胞）所識別。抗體中能識別抗原表位的區域叫做「互補位」或「抗體決定簇」（Paratope）。儘管通常抗原表位是指外來蛋白質等物質的其中一部分，但只要能被自身免疫系統所識別的表位，也被歸為抗原表位。

蛋白質抗原的表位根據它們的結構以及與互補位的交互作用，被分為構象表位Conformational epitopes和線性表位Linear epitopes這兩種類型。其中構象表位有抗原胺基酸序列中的不連續部分組成，因此互補位和抗原表位的交互作用是基於表面的三位特徵和形狀，或者是抗原的三級結構。大部分的抗原表位都屬於構象表位。與此相反，線性表位是由一段連續的抗原胺基酸序列構成，與抗原的交互作用的基礎是其一級結構。

抗原被抗体辨識或結合的地方叫抗原決定位antigenic determinant或epitope。

抗原上的決定位通常含6～8個胺基酸，它可以是三度空間的構造conformation structure，如抗体結合到蛋白質暴露在外面的位置，如果將蛋白質變性，另一種抗原決定位是二度空間的構造sequence determinant，T-細胞所辨認的抗原決定位，便是此種一連串胺基酸組成的抗原決定位，它和主要組織相容抗原（Major histocompatibility complex；MHC）上的class I/II在一起，而與T-細胞上的T-細胞受器（T cell receptor，TCR）結合，一個抗原通常有好幾個抗原決定位，構造越複雜，分子量越大，它的抗原決定位愈多。T-細胞和B-細胞辨認的抗原決定位可以是不同的。

APC：Antigen-presenting cell

An Antigen-presenting cell（APC）or Accessory cell is a cell that displays foreign antigens complexed with Major histocompatibility complexes（MHC's）on their surfaces. T-cells may recognize these complexes using their T-cell receptors（TCRs）. These cells process antigens and present them to T-cells.

APCs fall into two categories：professional or non-professional.

T cells cannot recognize, and therefore cannot respond to, 'free' antigen. T cells can only 'see' an antigen that has been processed and presented by cells via carrier molecules like MHC and CD1 molecules. Most cells in the body can present antigen to CD8+ T cells via MHC class I molecules and, thus, act as "APCs"; however, the term is often limited to specialized cells that can prime T cells（i.e., activate a T cell that has not been exposed to antigen, termed a naive T cell）. These cells, in general, express MHC class II as well as MHC class I molecules, and can stimulate CD4+（helper）cells as well as CD8+（cytotoxic）T cells, respectively.

Almost all nucleated cells express MHC class I receptors, including professional APCs. If a virus infects a macrophage or dendritic cell, it will try to promote its own destruction through cytotoxic T cells. However, dendritic cells can ingest viruses through pinocytosis and therefore activate the adaptive immune response to create antibodies for the virus through class II MHC receptors.

To help distinguish between the two types of APCs, those that express MHC class II molecules are often called professional antigen-presenting cells.

Concise review：Adipose-derived stem cells as a novel tool for future regenerative medicine.

Stem Cells. 2012 May;30(5):804-10. doi: 10.1002/stem.1076.

Mizuno H, Tobita M, Uysal AC.

Abstract

The potential use of stem cell-based therapies for the repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of diseases. The use of either embryonic stem cells (ESCs) or induced pluripotent stem cells in clinical situations is limited due to cell regulations and to technical and ethical considerations involved in the genetic manipulation of human ESCs, even though these cells are, theoretically, highly beneficial.

Mesenchymal stem cells seem to be an ideal population of stem cells for practical regenerative medicine, because they are not subjected to the same restrictions. In particular, large number of Adipose-derived stem cells (ASCs) can be easily harvested from adipose tissue. Furthermore, recent basic research and preclinical studies have revealed that the use of ASCs in regenerative medicine is not limited to Mesodermal tissue but extends to both Ectodermal and Endodermal tissues and organs, although ASCs originate from mesodermal lineages. Based on this background knowledge, the primary purpose of this concise review is to summarize and describe the underlying biology of ASCs and their proliferation and differentiation capacities, together with current preclinical and clinical data from a variety of medical fields regarding the use of ASCs in regenerative medicine. In addition, future directions for ASCs in terms of cell-based therapies and regenerative medicine are discussed.

Copyright © 2012 AlphaMed Press.

Monocytes can be used to generate Dendritic cells in vitro by adding Cytokines（Granulocyte Monocyte Colony Stimulating Factor；GMCSF）and IL-4.